Monomeric Fc Fusion Technology: An Approach to Create Long-Lasting Clotting Factors

SummaryBlood coagulation in a strain of rabbits designated as Watanabe heritable hyperlipidemic (WHHL) rabbits was examined. The activities of vitamin K-dependent clotting factors, contact factors and clotting factor VIII (F VIII) and the fibrinogen level were significantly higher in WHHL rabbits than in normolipidemic rabbits (all age groups). Values for vitamin Independent clotting factor were already higher at 2 months of age. Contact factors and fibrinogen levels increased age after 5 to 8 months. F VIII increased between 5 and 8 months and then decreased. At 2 months of age, WHHL rabbits were divided into two groups. Group A was fed standard rabbit chow and group B standard rabbit chow containing 1% probucol. Probucol prevented the progression of atherosclerosis in group B in the absence of a significant reduction in plasma cholesterol level. F VIII and fibrinogen levels were statistically decreased in all rabbits at all ages in group B (P<0.05). These differences in clotting factors between the two groups were most obvious at 8 months (P<0.02).We conclude that vitamin K-dependent clotting factors may increase with hyperlipemia and that increases in F VIII and fibrinogen may be closely related to the progression of throm- boatherosclerosis.

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Scanning Electron Microscopic and Electrophoretic Observations on Barium Sulphate Used to Adsorb Clotting Factors

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647879 ◽

1975 ◽

Vol 33 (02) ◽

pp. 256-270

Author(s):

R. M Howell ◽

S. L. M Deacon

Keyword(s):

Electron Microscopy ◽

Factor Xa ◽

Barium Sulphate ◽

High Density ◽

Low Density ◽

Factor X ◽

Clotting Factors ◽

Electron Microscopic ◽

Complementary Techniques ◽

Scanning Electron

SummaryElectron microscopy and particle electrophoresis were found to be complementary techniques with which to complete the physical data from an earlier study on barium sulphates used to adsorb clotting factors from serum. The differences revealed by scanning electron microscopy (S. E. M.) in the physical shape of low and high density grades of barium sulphate particles appear to be of greater significance than charge as expressed by electrophoretic mobility, in determining whether or not precursor or preformed factor Xa is eluted.This conclusion was based on the finding that at pH values close to 7, where the adsorption from serum occurs, all samples with the exception of natural barytes were uncharged. However as the high-density, or soil-grade, was found by S. E. M. to consist of large solid crystals it was suggested that this shape might induce activation of factor X as a result of partial denaturation and consequent unfolding of the adsorbed protein. In contrast, uptake of protein into the centre of the porous aggregates revealed by S. E. M. pictures of low-density or X-ray grade barium sulphate may afford protection against denaturation and exposure of the enzyme site.The porous nature of particles of low-density barium sulphate compared with the solid crystalline forms of other grades accounts not only for its lower bulk density but also for its greater surface/gram ratio which is reflected by an ability to adsorb more protein from serum.Neither technique produced evidence from any of the samples to indicate the presence of stabilising agents sometimes used to coat particles in barium meals.

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Recombinant Tissue Factor as Substitute for Conventional Thromboplastin in the Prothrombin Time Test

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648376 ◽

1992 ◽

Vol 67 (01) ◽

pp. 042-045 ◽

Cited By ~ 13

Author(s):

Armando Tripodi ◽

Arnaldo Arbini ◽

Veena Chantarangkul ◽

Pier Mannuccio Mannucci

Keyword(s):

Prothrombin Time ◽

Tissue Factor ◽

Oral Anticoagulant Therapy ◽

Clotting Factor ◽

Rabbit Brain ◽

Sensitivity Index ◽

Clotting Factors ◽

Wide Range ◽

Reference Preparation ◽

Time Test

SummaryRelipidated recombinant tissue factor (r-TF) has been assessed in comparison with conventional rabbit brain thromboplastin (Manchester Reagent) for its suitability for measurement of prothrombin time (PT). The International Sensitivity Index (ISI) of r-TF calibrated against the International Reference Preparation BCT/253 (human plain) was found to be 0.96 and 1.12 with instrumental and manual techniques. Our study of plasmas from patients with congenital deficiencies of clotting factors covering a wide range of severity demonstrates that r-TF is able to detect even minor deficiencies of factors involved in the extrinsic and common coagulation pathways. Patients with liver diseases were correctly diagnosed with a prevalence of abnormal results comparable for both reagents. Between-assay reproducibility expressed as coefficient of variation was 2.3 % and 3.9 % at normal and abnormal PT levels.In conclusion, our evaluation shows that relipidated r-TF possesses the necessary requisites of sensitivity, diagnostic accuracy and reproducibility which make it a suitable candidate for PT determination both for monitoring oral anticoagulant therapy and diagnosing congenital and acquired clotting factor deficiencies. Moreover, being a highly defined reagent it may constitute a step forward in the standardization of PT testing.

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Mutations which Introduce Free Cysteine Residues in the Gla-Domain of Vitamin K Dependent Proteins Result in the Formation of Complexes with α1-Microglobulin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650223 ◽

1996 ◽

Vol 75 (01) ◽

pp. 070-075 ◽

Cited By ~ 20

Author(s):

E G C Wojcik ◽

P Simioni ◽

M v d Berg ◽

A Girolami ◽

R M Bertina

Keyword(s):

Vitamin K ◽

Protein C ◽

Cysteine Residue ◽

Factor Ix ◽

Disulphide Bond ◽

Clotting Factors ◽

High Molecular Weight Complex ◽

Low Concentrations ◽

Gla Domain ◽

Formation Of Complexes

SummaryWe have previously described a genetic factor IX variant (Cys18→Arg) for which we demonstrated that it had formed a heterodimer with armicroglobulin through formation of a disulphide bond with the remaining free cysteine residue of the disrupted disulphide bond in the Gla-domain of factor IX. Recently, we observed a similar high molecular weight complex for a genetic protein C variant (Arg-1→Cys). Both the factor IX and the protein C variants have a defect in the calcium induced conformation. In this study we show that the aminoterminus of this protein C variant is prolonged with one amino acid, cysteine. This protein C variant, as well as protein C variants with Arg9→Cys and Ser12→Cys mutations which also carry a free cysteine residue, are shown to be present in plasma as a complex with α1-microglobulin. A prothrombin variant with a Tyr44→Cys mutation, had not formed such a complex. Furthermore, complexes between normal vitamin K-dependent clotting factors and α1-microglobulin were shown to be present in plasma at low concentrations. The data suggest that the presence of an unpaired cysteine residue in the propeptide or the N-terminal half of the Gla-domain has strongly promoted the formation of a complex with α1-microglobulin in the variants.

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Kinetic Aspects of the Interaction of Blood-Clotting Enzymes

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651250 ◽

1968 ◽

Vol 20 (01/02) ◽

pp. 078-087 ◽

Cited By ~ 34

Author(s):

H. C Hemker ◽

A. D Muller

Keyword(s):

Vitamin K ◽

Blood Clotting ◽

Experimental Results ◽

Factor X ◽

Clotting Factors ◽

Vitamin K Deficiency ◽

K Deficiency

SummaryPIVKA, the circulating anticoagulant protein found in vitamin K deficiency can, on kinetical grounds, be recognized as an analogue of factor X. The existence of analogues of other vitamin K-dependent clotting factors cannot be ruled out, but need not be assumed to explain the experimental results.

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Studies on the Interaction of Warfarin and Clofibrate in Man

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649370 ◽

1972 ◽

Vol 27 (02) ◽

pp. 309-318 ◽

Cited By ~ 2

Author(s):

R. A O’Reilly ◽

M. A Sahud ◽

A. J Robinson

Keyword(s):

Platelet Aggregation ◽

Oral Anticoagulant Therapy ◽

Adenosine Diphosphate ◽

Normal Subjects ◽

Term Therapy ◽

Rapid Decline ◽

Hemorrhagic Diathesis ◽

Platelet Adhesiveness ◽

Clotting Factors ◽

One Stage

SummaryTo evaluate the basis for the hemorrhagic diathesis associated with oral anticoagulant therapy plus the hypolipidemic agent clofibrate, this interaction was studied in 8 normal subjects. Administration of clofibrate 2.0 g/day alone for 14 days had no effect on the platelet count, bleeding time, platelet aggregation, plasma adenosine diphosphatase, platelet release of adenosine diphosphate and adenosine triphosphate, platelet-collagen adhesion, one-stage prothrombin time, or vitamin K-dependent clotting factors (II, VII, IX and X) but significantly reduced platelet adhesiveness and epinephrine-induced platelet aggregation. After addition of large single doses of sodium warfarin, 1.5 mg/kg body weight, to the clofibrate regimen, all values remained within the normal range, including platelet adhesiveness, except epinephrine-induced platelet aggregation. The one-stage prothrombin activity and clotting factors II and X were significantly lower with warfarin plus clofibrate than with warfarin alone, but the plasma level of warfarin was unchanged. Co-administration of sodium warfarin and clofibrate for 21 days augmented the hypoprothrombinemia observed in long-term therapy with warfarin alone but caused no significant change in the plasma warfarin level. It is concluded that the hemorrhagic complications of therapy with warfarin plus clofibrate result primarily from the more rapid decline in the activities of clotting factors II and X and perhaps also from the reduced platelet aggregation.

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Phospholipid-Protein Interactions in the Formation of Prothrombin Activator

Thrombosis and Haemostasis ◽

10.1055/s-0038-1654880 ◽

1965 ◽

Vol 14 (03/04) ◽

pp. 431-444 ◽

Cited By ~ 12

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.

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A Study of Three Cases of Familial Congenital Hypoprothrombinaemia (Factor II Deficiency)

Thrombosis and Haemostasis ◽

10.1055/s-0038-1654845 ◽

1964 ◽

Vol 11 (02) ◽

pp. 497-505 ◽

Cited By ~ 3

Author(s):

O de Bastos ◽

R. S Reno ◽

O. T Correa

Keyword(s):

Clotting Factors ◽

Factor Ii ◽

Group B ◽

Haemorrhagic Disease ◽

Rule Out

SummaryThree cases of true congenital hypoprothrombinaemia are presented.Studies were made to prove the deficiency of prothrombin in the patient’s plasma and to rule out deficiency of other clotting factors or the presence of abnormal anti-thrombin substances.All the patients were of group B.The results obtained confirm that the patients have a real prothrombin deficiency as the cause of a severe haemorrhagic disease.

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Intoxication with Phenprocoumon (Marcoumar) Pharmacokinetics and Side Effects

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653541 ◽

1969 ◽

Vol 21 (02) ◽

pp. 320-324 ◽

Cited By ~ 3

Author(s):

K Seiler ◽

F Duckert

Keyword(s):

Plasma Concentration ◽

Side Effects ◽

Half Life ◽

Plasma Proteins ◽

Repeated Administration ◽

High Plasma ◽

Vitamin K1 ◽

Clotting Factors ◽

Therapeutic Concentration ◽

High Plasma Concentration

SummaryA case of severe Marcoumar intoxication is described. Eleven hours after the intake a plasma concentration of 15.75 µg/ml was found which corresponds approximately to the 5-fold therapeutic concentration. Repeated administration of vitamin K1 made it possible to avoid extreme lowering of the activity of the clotting factors II, VII and X and to prevent bleeding. Side effects were not observed. The biologic half-life of Phenprocoumon has been found to be shortened at high plasma concentration (3.7 instead of 5.9 days). It is probable that in extreme concentration the drug is less strongly bound to the plasma proteins.

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The Reaction of Thiol Compounds with the Vitamin-K Dependent Clotting Factors

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653570 ◽

1969 ◽

Vol 21 (03) ◽

pp. 573-579 ◽

Cited By ~ 1

Author(s):

P Fantl

Keyword(s):

Prothrombin Time ◽

Vitamin K ◽

Anaerobic Conditions ◽

Clotting Factors ◽

Thiol Compounds ◽

Aerobic Conditions ◽

One Stage ◽

Factor Ii ◽

Ph Dependent ◽

The One

SummaryTreatment of human and dog oxalated plasma with 0.2 to 1.0 × 10−1 M 2.3-dithiopropanol (BAL) or dithiothreitol (DTT) at 2–4° C for 30 min results in the reduction of the vitamin-K dependent clotting factors II, VII, IX and X to the respective-SH derivatives. The reaction is pH dependent. Under aerobic conditions the delayed one stage prothrombin time can be partly reversed. Under anaerobic conditions a gradual prolongation of the one stage prothrombin time occurs without reversal.In very diluted plasma treated with the dithiols, prothrombin can be converted into thrombin if serum as source of active factors VII and X is added. In contrast SH factors VII, IX and X are inactive in the specific tests. Reoxidation to active factors II, VII, IX and X takes place during adsorption and elution of the SH derivatives. The experiments have indicated that not only factor II but also factors VII, IX and X have active-S-S-centres.

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