The Separation of Thromboplastin Formed from Pig’s Plasma in the Thromboplastin Generation Test

1962 ◽  
Vol 08 (03) ◽  
pp. 485-501
Author(s):  
M. J Cross
Keyword(s):  
Original System ◽  
Factor V ◽  
Generation System ◽  
Thromboplastic Activity ◽  
Generation Test

Summary1. Plasma thromboplastin has been formed from a mixture of pigs’ plasma, serum and platelets using a modification of the thromboplastin generation system of Biggs and Douglas (1953). The thromboplastic activity in the modified system was more stable than in the original system.2. A sediment with considerable thromboplastic activity has been obtained by centrifugation. This sediment was free of platelets and contained very little thrombin.3. The sediment when resuspended in buffer was fully active only in the presence of calcium and between pH 6.6 and 7.0. The activity slowly decreased at 0—4° C and rapidly at 65° C.4. The sediment rapidly converted prothrombin to thrombin in the absence of factor V.5. The activity of the sediment was unaffected when it was incubated with thrombin.

Action of a Proteolytic Enzyme Inhibitor on Blood Coagulation in Vitro

1967 ◽  
Vol 18 (01/02) ◽  
pp. 190-197 ◽  
Author(s):  
B Blombäck ◽  
Margareta Blombäck ◽  
P Olsson
Keyword(s):  
Blood Coagulation ◽  
Proteolytic Enzyme ◽  
Enzyme Inhibitor ◽  
Factor V ◽  
Generation System ◽  
Antifibrinolytic Agents ◽  
Coagulation Activity ◽  
Serum Factors ◽  
Proteolytic Enzyme Inhibitor

SummaryThe action of the kallikrein and trypsin inhibitor, Trasylol, has been studied in a three-stage thrombin generation system. It has been found that Trasylol inhibits one or several of the early reactions of blood coagulation. The inhibition of the early stages is dependent on the concentration of inhibitor, serum factors and factor VIII. The activation of factor V by tiger snake venom is not inhibited by Trasylol. The inhibition is most probably on one or several of the reactions preceding the factor V involved step.Some other known antifibrinolytic agents have also been tested for anti-coagulation activity.

Cleavage Requirements of Factor V in Tissue-factor Induced Thrombin Generation

1998 ◽  
Vol 80 (07) ◽  
pp. 92-98 ◽  
Author(s):  
Elisabeth Thorelli ◽  
Randal Kaufman ◽  
Björn Dahlbäck
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Lag Phase ◽  
Factor V ◽  
Relative Importance ◽  
Cleavage Sites ◽  
Wild Type ◽  
Generation System ◽  
Subsequent Phase ◽  
Prothrombinase Activity

SummaryFactor V (FV) activation is the result of cleavages at Arg709, Arg1018 and Arg1545 by thrombin or FXa. The relative importance of these cleavages in tissue factor (TF) induced thrombin generation in plasma and in a purified system was elucidated with recombinant FV in which the three sites had been eliminated one by one or in combinations. The mutants were analyzed with a clotting assay using FV-deficient plasma and in a TF induced thrombin generation system using plasma or purified components. Surprisingly, in the standard FV clotting assay, all mutants gave similar clotting activities and the thrombin generation curves obtained with wild-type and thrombin-resistant FV were similar. Differences in clotting activities and thrombin generation patterns between wild-type and thrombin-resistant FV were only observed when lower TF concentrations were used. The thrombin generation curve obtained in plasma containing wt FV was characterized by a short lag phase and a subsequent phase of rapid thrombin generation (propagation phase). The Arg709 to Gln mutation yielded a slightly prolonged lag phase and the rate of thrombin generation during the propagation phase was approximately 5-fold lower than that observed with wt FV. The Arg1018 to Ile mutation only slightly affected the thrombin generation curve, whereas the Arg1545 to Gln mutation yielded a prolonged lag phase and decreased maximum thrombin activity. Thrombin-resistant FV (mutated at all three sites) yielded a prolonged lag phase and poor thrombin generation during the propagation phase. The purified system further demonstrated the importance of the three cleavage sites for rapid and sustained thrombin generation. The results demonstrate that cleavages at positions 709, 1018 and 1545 are not required for assembly of a FXa-FV complex expressing low but significant prothrombinase activity but that all three sites in different ways are important for the creation of a FVa which maximally supports the FXa-mediated activation of prothrombin.

scholarly journals A Simple Method for the Assay of Factor VIII

Blood ◽  
1960 ◽  
Vol 15 (5) ◽  
pp. 637-645 ◽  
Author(s):  
LUIS JOSÉ BERGNA
Keyword(s):  
Factor Viii ◽  
Bovine Serum ◽  
Factor V ◽  
Normal Human Plasma ◽  
Factor Viii Activity ◽  
Simple Method ◽  
Serum Factors ◽  
Standard Normal ◽  
Normal Human ◽  
Generation Test

Abstract A simple method for the assay of factor VIII activity, based on the thromboplastin generation test of Biggs and Douglas, has been described. Bovine serum is used as a source of factor V and serum factors, and plasma from a hemophiliac is not required. The concentration of factor VIII of the test plasma is compared to that of a standard normal human plasma. The results obtained with the method have been fairly reproducible.

Thrombin generation in first-degree relatives of patients with venous thromboembolism who have factor V Leiden

2008 ◽  
Vol 99 (01) ◽  
pp. 223-228 ◽  
Author(s):  
Jèrôme Duchemin ◽  
Christophe Leroyer ◽  
Bènèdicte Delahousse ◽  
Jean François Abgrall ◽  
Dominique Mottier ◽  
...  
Keyword(s):  
Venous Thromboembolism ◽  
Odds Ratio ◽  
Thrombin Generation ◽  
Adjusted Odds Ratio ◽  
Factor V Leiden ◽  
Factor V ◽  
Oestrogen Therapy ◽  
Prothrombotic State ◽  
First Degree Relatives ◽  
Generation Test

SummaryThe thrombin generation test appears to be a highly sensitive and specific test in the detection of thrombophilia in patients with venous thromboembolism. We aimed to determine the accuracy of the thrombin generation test to detect factorV Leiden and/or other prothrombotic states in first-degree relatives of patients with venous thromboembolism and factor V Leiden. Sixty-two first-degree relatives of 21 index cases were tested for factor V Leiden, the G20210A prothrombin gene mutation and thrombin generation. Information about oestrogen therapy and previousVTE was also collected. The normalized Thrombomodulin sensitivity ratio (n-TMsr) was defined as the ratio of endogenous thrombin potential determined in the presence and absence of thrombomodulin which was normalized against the same ratio determined in normal control plasma. The mean n-TMsr was 1.37 (± 0.33) in the 45 relatives with one or more prothrombotic state (factor V Leiden, G20210A prothrombin mutation, oestrogen therapy or hormonal therapy) and 1.02 (± 0.34) in the 17 relatives without prothrombotic state (p = 0.001). The positive predictive value was 90.3 (95%CI, 73.1 – 97.4). In relatives with an abnormal n-TMsr, the adjusted odds ratio for having a prothrombotic state was 8.3 (95%CI, 1.9 – 36.9) and the adjusted odds ratio for having the factor V Leiden was 14.3 (95%CI, 2.9 – 71.2).An abnormal thrombin generation test appears highly predictive for having factor V Leiden and/or other prothrombotic states in first-degree relatives of patients with venous thromboembolism and factor V Leiden.

Modified Tests for Measuring the Thrombogenic Potential of Factor IX Concentrates.

1979 ◽  
Author(s):  
Sarah M. Middleton ◽  
Jessie T. Douglas ◽  
C.D. Forbes ◽  
C.R.M. Prentice
Keyword(s):  
Factor Xa ◽  
Activated Partial Thromboplastin Time ◽  
Factor Ix ◽  
Procoagulant Activity ◽  
In Vitro Tests ◽  
Factor V ◽  
Thrombogenic Potential ◽  
Sepharose 4B ◽  
Generation Test

In vitro tests for screening potential thrombogenicity of factor IX concentrates are unsatisfactory as it has been shown that the non-activated partial thromboplastin time (NAPTT) and the TGt50 (reflecting thrombin generation by the concentrate after recalcification} do not correlate with each other. We have modified the TGt50 by addition of optimum concentrations of factor V and phospholipid, and compared it with the NAPTT and factor Xa generation test using the chromogenic substrate S2222. The modified TGtSO correlates with both the NAPTT and the factor Xa generation test suggesting that these tests are measuring the same entity. Chromatography of concentrates on sepharose 4B indicates that the high MW void volume material has procoagulant activity as measured by the unmodified TGt50 whilst the retained volume he procoagulant activity as measured by the TGt50 and the NAPTT. When, however, phospholipid is added to the unmodified TG150 the activity of the high MW component is lost and only that of the retained volume is still present. The data suggests that the modified TGt50 and the NAPTT measure the same procoagulant activity in these concentrates.

Thromboplastic Activity of Split Products of Phosphatidyl Ethanolamine

1960 ◽  
Vol 04 (02) ◽  
pp. 283-288 ◽  
Author(s):  
Karl H. Slotta ◽  
Erwin Deutsch
Keyword(s):  
Phosphatidyl Ethanolamine ◽  
Stable Suspension ◽  
Thromboplastic Activity ◽  
Generation Test

SummaryPure synthetic L-α-(dioleoyl) PE is inactive as a platelet substitute in the thromboplastin generation test, even when in a stable suspension. Under humid conditions it is partly hydrolyzed. Among the split products lyso PE and phosphoryl ethanolamine were identified. The resulting mixture has thromboplastin generating activity. This thromboplastin is formed slowly, but it is stable over longer periods of time than thromboplastins formed from PS + PC or PS + PE mixtures, which are rapidly generated and lose activity faster.

scholarly journals The Blood Clotting Properties of Rabbit Peritoneal Leukocytes in Vitro

1967 ◽  
Vol 17 (01/02) ◽  
pp. 222-236 ◽  
Author(s):  
S. I Rapaport ◽  
P. F Hjort
Keyword(s):  
Blood Clotting ◽  
Anticoagulant Activity ◽  
In Vitro Study ◽  
Factor V ◽  
Clotting Factors ◽  
Clotting System ◽  
Procoagulant Effect ◽  
Peritoneal Leukocytes ◽  
Thromboplastic Activity

SummaryA systematic in vitro study has been carried out of the blood clotting properties of rabbit peritoneal leukocytes. Rabbit heterophils have been shown to possess weak but definite tissue thromboplastic activity. They also contain an anticoagulant activity which is active in the intrinsic clotting system, in the extrinsic clotting system with brain thromboplastin, and in clotting systems with Russell’s viper venom. When the thromboplastic activity of the white cell itself initiates clotting, the anticoagulant is much less effective and the procoagulant effect of the WBC suspension predominates. Rabbit heterophils do not bind plasma clotting factors, do not activative factor V, and, under our conditions, do not precipitate fibrinogen from normal plasma, from plasma exposed to traces of thrombin in vitro, or from plasma of rabbits given endotoxin.The relation of these findings to the role of the granulocyte in the pathogenesis of the generalized Shwartzman reaction has been discussed.

PHOSPHOLIPIDS: II. A CORRELATION OF CHEMICAL STRUCTURE WITH THROMBOPLASTIC ACTIVITY

1965 ◽  
Vol 43 (9) ◽  
pp. 1465-1470 ◽  
Author(s):  
P. J. Grisdale ◽  
A. Okany
Keyword(s):  
Fatty Acid ◽  
Chain Length ◽  
Chemical Structure ◽  
Fatty Ester ◽  
Two Stage ◽  
Fatty Acid Chain Length ◽  
Fatty Acid Chain ◽  
Thromboplastic Activity ◽  
Generation Test

A series of synthetic phosphatidylserines containing saturated fatty ester residues (C6 to C18) has been synthesized. These compounds have been studied in combination with natural phosphatidylethanolamine in the two-stage thromboplastin-generation test. The observed activity of these mixtures depends on the fatty acid chain length of the phosphatidylserine. These variations are accounted for in terms of the micelle theory of thromboplastic activity.

scholarly journals Platelets and Platelet Phosphatides in Uremia

Blood ◽  
1960 ◽  
Vol 16 (4) ◽  
pp. 1439-1446 ◽  
Author(s):  
GERALD ALTSCHULER ◽  
AARON J. MARCUS ◽  
HARRIS L. ULLMAN
Keyword(s):  
Platelet Count ◽  
Prothrombin Time ◽  
Silicic Acid ◽  
Chromatographic Analysis ◽  
Normal Platelet ◽  
Chromatographic Pattern ◽  
Thromboplastic Activity ◽  
Phase Platelet ◽  
Generation Test ◽  
Column Chromatographic

Abstract 1. The platelets of 20 uremic subjects were studied, utilizing the following procedures; phase platelet count, serum prothrombin time, thromboplastin generation test with evaluation of plasma, serum, and platelet reagents. 2. Platelet phospholipids from these subjects were used as reagents in the thromboplastin generation test and examined by means of silicic acid paper and column chromatography. 3. Thrombocytopenia was the most common abnormality encountered and was associated with either normal or abnormal prothrombin consumption. Some patients demonstrated defective prothrombin utilization despite normal platelet counts, but their platelets had normal thromboplastic activity, as did those of all patients studied. 4. The paper chromatographic pattern of the phosphatides in all subjects was the same as that reported for normal platelets. Similar results were obtained on column chromatographic analysis of a pooled extract from nine of the uremic patients. 5. On the basis of these studies it was not possible to demonstrate a qualitative platelet defect in uremia.

Routine Factor V Leiden Testing Discouraged

2008 ◽  
Vol 41 (7) ◽  
pp. 26-27
Author(s):  
JANE SALODOF MACNEIL
Keyword(s):  
Factor V Leiden ◽  
Factor V
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