scholarly journals The coagulant-active phospholipid content is a major determinant of in vivo thrombogenicity of prothrombin complex (Factor IX) concentrates in rabbits

Blood ◽  
1982 ◽  
Vol 59 (2) ◽  
pp. 401-407 ◽  
Author(s):  
AR Giles ◽  
ME Nesheim ◽  
H Hoogendoorn ◽  
PB Tracy ◽  
KG Mann
Keyword(s):  
Factor Xa ◽  
Lipid Vesicles ◽  
Factor Ix ◽  
Major Determinant ◽  
Phospholipid Content ◽  
Clotting Factors ◽  
Prothrombin Complex Concentrates ◽  
Vitro Assay

Abstract In vitro evaluation of prothrombin complex concentrates in a thrombin generation assay, using DAPA and purified components of the prothrombinase complex, demonstrated significant levels of coagulant- active “phospholipid replacing” activity. Quantification of this activity showed a significant correlation (r = 0.8747, p less than 0.01) with thrombogenicity measured in vivo in a stasis model in rabbits. Extracted lipid material retained full phospholipid replacing activity in the vitro assay. Thin-layer chromatographic characterization confirmed the presence of phospholipids with known coagulant activity in vitro. In vivo, the extracted material was nonthrombogenic but augmented the thrombogenicity of purified factor Xa. Substitution of a synthetic coagulant-active phospholipid (phosphatidylcholine-phosphatidylserine lipid vesicles) for the extracted phospholipid produced a similar augmentation of a factor-Xa- induced thrombogenicity in vivo. It is concluded that the coagulant- active phospholipid content of prothrombin complex concentrates is a major determinant of thrombogenicity but requires the presence of activated clotting factors for its expression in vivo.

scholarly journals The coagulant-active phospholipid content is a major determinant of in vivo thrombogenicity of prothrombin complex (Factor IX) concentrates in rabbits

Blood ◽  
1982 ◽  
Vol 59 (2) ◽  
pp. 401-407 ◽  
Author(s):  
AR Giles ◽  
ME Nesheim ◽  
H Hoogendoorn ◽  
PB Tracy ◽  
KG Mann
Keyword(s):  
Factor Xa ◽  
Lipid Vesicles ◽  
Factor Ix ◽  
Major Determinant ◽  
Phospholipid Content ◽  
Clotting Factors ◽  
Prothrombin Complex Concentrates ◽  
Vitro Assay

In vitro evaluation of prothrombin complex concentrates in a thrombin generation assay, using DAPA and purified components of the prothrombinase complex, demonstrated significant levels of coagulant- active “phospholipid replacing” activity. Quantification of this activity showed a significant correlation (r = 0.8747, p less than 0.01) with thrombogenicity measured in vivo in a stasis model in rabbits. Extracted lipid material retained full phospholipid replacing activity in the vitro assay. Thin-layer chromatographic characterization confirmed the presence of phospholipids with known coagulant activity in vitro. In vivo, the extracted material was nonthrombogenic but augmented the thrombogenicity of purified factor Xa. Substitution of a synthetic coagulant-active phospholipid (phosphatidylcholine-phosphatidylserine lipid vesicles) for the extracted phospholipid produced a similar augmentation of a factor-Xa- induced thrombogenicity in vivo. It is concluded that the coagulant- active phospholipid content of prothrombin complex concentrates is a major determinant of thrombogenicity but requires the presence of activated clotting factors for its expression in vivo.

scholarly journals Factor XI contributes to thrombin generation in the absence of factor XII

Blood ◽  
2009 ◽  
Vol 114 (2) ◽  
pp. 452-458 ◽  
Author(s):  
Dmitri V. Kravtsov ◽  
Anton Matafonov ◽  
Erik I. Tucker ◽  
Mao-fu Sun ◽  
Peter N. Walsh ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Factor Ix ◽  
Factor Xii ◽  
Factor Xi ◽  
Factor Xii Deficiency ◽  
Low Concentrations

Abstract During surface-initiated blood coagulation in vitro, activated factor XII (fXIIa) converts factor XI (fXI) to fXIa. Whereas fXI deficiency is associated with a hemorrhagic disorder, factor XII deficiency is not, suggesting that fXI can be activated by other mechanisms in vivo. Thrombin activates fXI, and several studies suggest that fXI promotes coagulation independent of fXII. However, a recent study failed to find evidence for fXII-independent activation of fXI in plasma. Using plasma in which fXII is either inhibited or absent, we show that fXI contributes to plasma thrombin generation when coagulation is initiated with low concentrations of tissue factor, factor Xa, or α-thrombin. The results could not be accounted for by fXIa contamination of the plasma systems. Replacing fXI with recombinant fXI that activates factor IX poorly, or fXI that is activated poorly by thrombin, reduced thrombin generation. An antibody that blocks fXIa activation of factor IX reduced thrombin generation; however, an antibody that specifically interferes with fXI activation by fXIIa did not. The results support a model in which fXI is activated by thrombin or another protease generated early in coagulation, with the resulting fXIa contributing to sustained thrombin generation through activation of factor IX.

The Fibrinolytic Potential of the Normal Primate following the Generation of Thrombin In Vivo

1990 ◽  
Vol 63 (03) ◽  
pp. 476-481 ◽  
Author(s):  
Alan R Giles ◽  
Michael E Nesheim ◽  
Steven W Herring ◽  
Hugh Hoogendoorn ◽  
David C Stump ◽  
...  
Keyword(s):  
Time Course ◽  
Factor Xa ◽  
Lipid Vesicles ◽  
Experimental Period ◽  
In Vivo Studies ◽  
Full Time ◽  
Primate Model ◽  
Fibrinolytic Potential

SummaryParameters of the fibrinolytic system were studied in a primate model where the generation of thrombin was promoted in vivo. The procoagulant stimulus used was a combination of human factor Xa in combination with phosphatidylcholine/phos-phatidylserine lipid vesicles (PCPS) as the source of coagulant active phospholipid. The dosage of each component was formulated to provide a gradation of thrombin generating potential assessed prior to in vivo study in an in vitro clotting assay. These ranged from 25.25 - 36.60 pMole/kg (factor Xa) and 18.85 - 56.30 nMole/kg (PCPS). In each case, the ratio of the dose of factor Xa/PCPS was maintained at 0.65 (pMole factor Xa/ nMole PCPS). Individual dosage combinations producing recalcification clotting times in vitro of 15, 20, 25 and 30 s were used in detailed in vivo studies. Previous studies in dogs had confirmed the thrombin generating potential of factor Xa/PCPS infusions and demonstrated an associated activation ot protein C and increased fibrinolytic activity. This has now been extensively characterized in the chimpanzee as follows: 10 min after the infusion of the highest dose (36.6 pMole factor Xa/56.3 nMole PCPS kg bodyweight), the level of circulating t-PA had risen to 900 ng/ml (antigen), 885 IU/ml (functional). Dosage was observed with the lowest dose of 12.25 pMole factor Xa and 18.85 nMole PCPS being associated with relatively minor increases in circulating t-PA activity. There were no changes in u-PA at any dosage during the full time course of the experimental period (90 min). Plasminogen activation was also apparent with alpha-2 antiplasmin levels falling to 30 - 40% of pre-infusion levels at the highest dosages. There was also a significant consumption of fibrinogen and evidence of active fibrinolysis manifested by major increases in the levels of FDP, D-dimer and B-beta 1-42. The data strongly suggested that this was predominantly fibrinolysis rather than fibrinogenolysis and that the fibrinolytic response observed resulted from a major release of t-PA from available stores consequent to thrombin generation and presumably subsequent fibrin generation. These data illustrate the enormous fibrinolytic potential of the intact normal primate and may provide a model for study of the mechanism(s) by which the regulation of t-PA availability can be up- or down-regulated in health and disease.

Measurement of Activated Factor IX in Factor IX Concentrates: Correlation with In Vivo Thrombogenicity

1995 ◽  
Vol 73 (04) ◽  
pp. 675-679 ◽  
Author(s):  
Elaine Gray ◽  
Jill Tubbs ◽  
S Thomas ◽  
A Oates ◽  
M Boisclair ◽  
...  
Keyword(s):  
Significant Positive Correlation ◽  
Factor Ix ◽  
In Vitro Tests ◽  
Prothrombin Complex Concentrates ◽  
Thrombotic Risk ◽  
Chromogenic Assay ◽  
The Mean ◽  
Thrombogenic Potential

SummaryCurrent in vitro tests for thrombogenicity of FIX concentrates used for prothrombin complex concentrates (PCCs), are of little value when applied to high purity FIX (HP FIXs). In the present study, we have developed a chromogenic assay for activated FIX (FIXa) and evaluated its ability to predict in vivo thrombogenic potential of HP FIXs in a modified Wessler stasis model. Among the HP FIXs, only 1 out of 7 products had no detectable FIXa; this product also showed no in vivo thrombogenicity. In the other 6 products, FIXa content ranged from 0.15–1.2 U/1000 iu FIX, and all showed some evidence of in vivo thrombogenicity, with mean thrombus scores ranging from 0.25–4. There was a significant positive correlation (r = 0.55, p <0.02) between FIXa levels and in vivo thrombogenicity of HP FIXs. NAPTT data were not significantly correlated with the in vivo results and the TFCT also showed no direct correlation with the mean thrombus score. These results indicate that HP FIXs may still carry a small residual thrombotic risk and measurement of FIXa content of these products may be a better predictor of thrombogenicity than the current in vitro tests.

Feiba And Autoplex: A Comparison Of These Two Activated Prothrombin Complex Concentrates

1981 ◽  
Author(s):  
E Lechler ◽  
B Eggeling ◽  
D Meyer-Börnecke ◽  
H Stoy
Keyword(s):  
Hemophilia A ◽  
Gel Filtration ◽  
Activated Partial Thromboplastin Time ◽  
Factor Ix ◽  
Prothrombin Complex Concentrates ◽  
Minor Degree ◽  
Activated Prothrombin Complex Concentrates ◽  
A Minor

These activated concentrates are used for the treatment of patients with factor VIII inhibitors . Both shorten the activated and non-activated partial thromboplastin time of inhibitor plasma and hemophilia A plasma in vitro. They do not or only to a minor degree improve the prothrombin consumption of hemophilia A plasma in vitro. In gel filtration of AUTOPLEX the activity which shortens the PTT of hemophilia A plasma eluted in a volume higher than that of the nonactivated factors of the prothrombin complex and contains activated factor IX. The activity of FEIBA elutes at a lower filtration volume in a rather broad peak together with the factors of the the non-activated prothrombin complex. BaSO4- adsorbed plasma and purified antithrombin (Behring) abolish the activity of AUTOPLEX more readily than of FEIBA. Both concentrates have only a low amidolytic effect (S 2222) and are not inhibited with SBTI and PMSF. In the crossed two-dimensional immunelectrophoresis with heparin in the agarose of the first dimension and anti-antithrombin (Behring) in the agarose of the second dimension (method of Sas), a mixture of AUTOPLEX and antithrombin results into a two peak precipitation of antithrombin, whereas with FEIBA a broadened intermediate peak develops. In vivo both concentrates do not improve the prothrombin consumption and AUTOPLEX shortens the PTT for at least 90 minutes. In summary, these two concentrates differ considerably.

scholarly journals Sac1p mediates the adenosine triphosphate transport into yeast endoplasmic reticulum that is required for protein translocation.

1995 ◽  
Vol 131 (6) ◽  
pp. 1377-1386 ◽  
Author(s):  
P Mayinger ◽  
V A Bankaitis ◽  
D I Meyer
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Protein A ◽  
Lipid Vesicles ◽  
Secretory Proteins ◽  
Transport Activity ◽  
Vitro Assay ◽  
Microsomal Membranes

Protein translocation into the yeast endoplasmic reticulum requires the transport of ATP into the lumen of this organelle. Microsomal ATP transport activity was reconstituted into proteoliposomes to characterize and identify the transporter protein. A polypeptide was purified whose partial amino acid sequence demonstrated its identity to the product of the SAC1 gene. Accordingly, microsomal membranes isolated from strains harboring a deletion in the SAC1 gene (sac1 delta) were found to be deficient in ATP-transporting activity as well as severely compromised in their ability to translocate nascent prepro-alpha-factor and preprocarboxypeptidase Y. Proteins isolated from the microsomal membranes of a sac1 delta strain were incapable of stimulating ATP transport when reconstituted into the in vitro assay system. When immunopurified to homogeneity and incorporated into artificial lipid vesicles, Sac1p was shown to reconstitute ATP transport activity. Consistent with the requirement for ATP in the lumen of the ER to achieve the correct folding of secretory proteins, the sac1 delta strain was shown to have a severe defect in transport of procarboxypeptidase Y out of the ER and into the Golgi complex in vivo. The collective data indicate an intimate role for Sac1p in the transport of ATP into the ER lumen.

scholarly journals Bidirectional FtsZ filament treadmilling transforms lipid membranes via torsional stress

2019 ◽  
Author(s):  
Diego A. Ramirez-Diaz ◽  
Adrian Merino-Salomon ◽  
Fabian Meyer ◽  
Michael Heymann ◽  
German Rivas ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Lipid Membranes ◽  
Force Production ◽  
Lipid Vesicles ◽  
Torsional Stress ◽  
Bacterial Cell Division ◽  
Giant Vesicles ◽  
Vitro Assay

AbstractFtsZ is a key component in bacterial cell division, being the primary protein of the presumably contractile Z ring. In vivo and in vitro, it shows two distinctive features that could so far however not be mechanistically linked: self-organization into directionally treadmilling vortices on solid supported membranes, and shape deformation of flexible liposomes. In cells, circumferential treadmilling of FtsZ was shown to recruit septum-building enzymes, but an active force production remains elusive. To gain mechanistic understanding of FtsZ dependent membrane deformations and constriction, we designed an in vitro assay based on soft lipid tubes pulled from FtsZ decorated giant lipid vesicles (GUVs) by optical tweezers. FtsZ actively transformed these tubes into spring-like structures, where GTPase activity promoted spring compression. Operating the optical tweezers in lateral vibration mode and assigning spring constants to FtsZ coated tubes, we found that FtsZ rings indeed exerts 0.14 – 1.09 pN forces upon GTP hydrolysis, through torsional stress induced by bidirectional treadmilling. These directional forces could further be demonstrated to induce membrane budding with constricting necks on both, giant vesicles and E.coli cells devoid of their cell walls.

scholarly journals Characterization of Factor VIII-Inhibitor By-Passing Activity (FEIBA)

1977 ◽  
Author(s):  
K. Hess ◽  
N. Shih ◽  
G. Tishkoff
Keyword(s):  
Factor Viii ◽  
Factor Xa ◽  
Factor Ix ◽  
Coagulation Cascade ◽  
Factor Viii Inhibitor ◽  
Factor X ◽  
Clotting Factors ◽  
Human Factor Ix ◽  
Viii Inhibitor

In an attempt to identify the thrombogenic factor in human factor IX concentrates, we have studied the role of trace quantities of activated clotting factors employing an assay that compares the Factor VIII-like activity of IX concentrates with the ability of these products to restore to normal the abnormal activated partial thromboplastin time (APTT) of Factor VIII inhibitor plasma after 1 minute and 40 minute incubation. A coagulant activity (FEIBA) was evident when partially purified Factors X and II were combined in vitro. Factor Xa (4 × 10-4 u) plus Factor II gave negative results. Factor IIa (5.5 × 10-2u), when combined with Factor X, generated FEIBA. Activated clotting factors (Xa, IIa) when tested alone, at comparable levels, were devoid of FEIBA. Our results suggest a mechanism, distinct from activated clotting factors, that can effectively by-pass the Factor VIII defect in the coagulation cascade. The proposed mechanism appears to also by-pass the normal inhibitory properties (i.e., antithrombin III) of human blood.

A Distinction between the Role of Precursor and Activated Forms of Clotting Factors in the Genesis of Stasis Thrombi

1967 ◽  
Vol 18 (01/02) ◽  
pp. 012-023 ◽  
Author(s):  
S Wessler ◽  
E. T Yin ◽  
L. W Gaston ◽  
Iona Nicol
Keyword(s):  
Human Serum ◽  
Factor Ix ◽  
In Vivo Studies ◽  
Factor Vii ◽  
Factor X ◽  
Clotting Factors ◽  
Insight Into

Summary1. In vivo studies in rabbits have shown that the liver diminishes the thrombo-genicity of infused human serum.2. In vitro rabbit liver perfusions with human serum have demonstrated the loss of serum thrombogenicity within 5 min after the onset of the perfusion. Associated with this loss of thrombotic capacity is a marked decrease in the activation product (AP) and labile factor IX (PPA) activity in the infused serum.3. The liver appears to have the capacity to discriminate between circulating activated clotting activities such as AP and PPA and inactive procoagulants such as stable or genuine factor IX, factor VII and factor X. The latter are not cleared from the circulation by the liver.4. These studies provide some insight into the mechanism whereby circulating activated clotting activities and retarded blood flow predispose to thrombosis.

In Vivo Evaluation Of The Antithrombotic Effects Of Some Polysaccharides Of Ultra Low Molecular Weight

1981 ◽  
Author(s):  
J Fareed ◽  
H L Messmore ◽  
J Choay ◽  
C Lormeau ◽  
M Petitou ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Serine Proteases ◽  
Factor Xa ◽  
Low Molecular Weight ◽  
Thrombin Time ◽  
Prothrombin Complex Concentrates ◽  
In Vivo Evaluation ◽  
Activated Prothrombin Complex Concentrates

Ultra low molecular weight saccharide fragments (ULMFs) have been obtained from porcine mucosal heparin (PMH) by extraction (e-ULMF) and by bacterial heparinase depolymerization (h-ULMF-8) processes. Both fragments showed a strong anti Xa activity (>2000 u/mg units, Yin and Wessler, J. Lab. Clin. Med. 81, 298, 1973) and possess relatively weak potencies in the US Pharmacoepial (<40 USP u/mg) and other conventional coagulant assays (activated and non activated partial thromboplastin time, thrombin time and whole blood activated recalcification times). Since ULMFs showed a strong anti Xa activity, we evaluated their antithrombotic actions in a modified stasis-thrombosis model (Wessler et. al. J. App. Phys. 14, 943, 1959) challenging the animals with various thrombogenic stimuli; activated and non activated prothrombin complex concentrates, factor Xa concentrates and human serum. h-ULMF-8 at dosage <0.125 mg/kg (<250 Anti Xa u/kg) IV and <1.0 mg/kg (>2000 anti Xa u/kg) SC completely protected the thrombogenic effects of various thrombogenic agents, whereas PMH at these dosages failed to produce any protection in pre and post treatment regiments. Similar studies with e-ULMF showed protection, however, the antithrombotic responses varied among animals. In vitro supplementation of heparin fragments at 5 times the concentration which protected animals against the thrombogenic effects of activated prothrombin complex concentrates failed to produce any elevation of prothrombin time, partial thromboplastin times, thrombin time and other coagulant assays. Our studies suggest that ULMFs are potent antithrombotic agents and may exert their effects involving multiple sites and primarily inhibiting the Xa and the non-thrombin serine proteases formed during activated states.

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