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WO1999066939A1 - Preparations de complexes thromboplastine tissulaire-vesicule phospholipidique, leurs procedes de fabrication et d'utilisation - Google Patents

Preparations de complexes thromboplastine tissulaire-vesicule phospholipidique, leurs procedes de fabrication et d'utilisation Download PDF

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Publication number
WO1999066939A1
WO1999066939A1 PCT/US1999/014183 US9914183W WO9966939A1 WO 1999066939 A1 WO1999066939 A1 WO 1999066939A1 US 9914183 W US9914183 W US 9914183W WO 9966939 A1 WO9966939 A1 WO 9966939A1
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Prior art keywords
thrombin
complex
phospholipid
thrombomodulin
tissue factor
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PCT/US1999/014183
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English (en)
Inventor
Victor J. Marder
Katalin Varadi
Juergen Siekmann
Peter L. Turecek
Hans-Peter Schwarz
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Marder Victor J
Katalin Varadi
Juergen Siekmann
Turecek Peter L
Schwarz Hans Peter
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Application filed by Marder Victor J, Katalin Varadi, Juergen Siekmann, Turecek Peter L, Schwarz Hans Peter filed Critical Marder Victor J
Priority to AU47093/99A priority Critical patent/AU4709399A/en
Publication of WO1999066939A1 publication Critical patent/WO1999066939A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/745—Assays involving non-enzymic blood coagulation factors
    • G01N2333/7454—Tissue factor (tissue thromboplastin, Factor III)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/745—Assays involving non-enzymic blood coagulation factors
    • G01N2333/7456—Factor V
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96433—Serine endopeptidases (3.4.21)
    • G01N2333/96441—Serine endopeptidases (3.4.21) with definite EC number
    • G01N2333/96461—Protein C (3.4.21.69)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/974—Thrombin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2405/00—Assays, e.g. immunoassays or enzyme assays, involving lipids
    • G01N2405/04—Phospholipids, i.e. phosphoglycerides

Definitions

  • the present invention relates to lipid vesicle-tissue factor complexes, preferably phospholipid-tissue factor (PL-TF) complexes (which optionally include thrombomodulin) and preparations containing such complexes.
  • PL-TF phospholipid-tissue factor
  • the complexes according to the invention can be used as a new assay system for simultaneous measurement of thrombin and APC generation in plasma or blood, among other things.
  • the physiologic response to vascular trauma is a finely ordered cascade of cellular and molecular events culminating in hemostatic plug formation.
  • Natural inhibitory mechanisms most notably the endothelial-associated thrombomodulin/protein C/protein S pathway, provide built-in checks on hemostasis, especially to limit its location to the injury site. Disruption of the inhibitory mechanisms allows the process to occur at inappropriate sites, which might lead to venous thrombosis following otherwise innocuous venous stasis or to widespread microvascular occlusion and purpura fulminans, which may occur after inflammatory events.
  • thrombomodulin TM
  • tissue factor when in high concentrations and surface density on a phospholipid vesicle exerts complementary effects of promoting the regular procoagulant cascade while inhibiting the effects of the protein C pathway.
  • Thrombomodulin in conjunction with tissue factor also can be employed according to the invention.
  • a complex comprised of tissue factor bound to a lipid vesicle, preferably a phospholipid vesicle.
  • Thrombomodulin also can be bound to the phospholipid vesicle to form a complex comprised of a phospholipid vesicle, tissue factor and thrombomodulin.
  • the phospholipid vesicle can comprise l,2-dioleyl-sn-glycero-3-phosphocholine (DOPC) and l-palmitoyl-2-oleyl-sn-glycero-3-phosphoserine (POPS), and have a diameter of about 200 nm to about 300 nm, for example (vesicles outside of this range can be employed and can be readily attained by the skilled person in view of the teachings contained herein).
  • the vesicles can be unilamellar, and the complex can comprise a multiple number of molecules of tissue factor (preferably 10 or more) bound to the phospholipid vesicle. In certain embodiments, the complex also can comprise thrombomodulin bound to the phospholipid vesicle.
  • kits for evaluating blood or plasma samples comprising a complex comprised of tissue factor bound to a phospholipid vesicle.
  • the phospholipid vesicle can comprise l,2-dioleyl-sn-glycero-3-phosphocholine (DOPC) and l-palmitoyl-2-oleyl-sn- glycero-3-phosphoserine (POPS), and have a diameter of about 200 nm to about 300 nm, for example (vesicles outside of this range can be employed and can be readily attained by the skilled person in view of the teachings contained herein).
  • DOPC dioleyl-sn-glycero-3-phosphocholine
  • POPS l-palmitoyl-2-oleyl-sn- glycero-3-phosphoserine
  • the vesicles can be unilamellar, and the complex can comprise a multiple number of molecules of tissue factor (preferably 10 or more) bound to the phospholipid vesicle. In certain embodiments, the complex also can comprise thrombomodulin bound to the phospholipid vesicle.
  • methods of assaying active blood proteins in a blood or plasma sample from a test subject comprising contacting the blood or plasma sample with a complex comprising tissue factor bound to a phospholipid vesicle, and determining the amount of active blood proteins in the sample from a test subject (such as a person who possesses a lupus inhibitor and/or as an approach to diagnose or characterize pathological thrombosis), wherein the active blood proteins are at least one selected from the group consisting of activated protein C and thrombin.
  • the contacting step can include contact with thrombomodulin, which along with thrombin is involved in the activation of protein C, and can be used to determine the presence of activated protein C. If thrombomodulin is not part of the contacting step or the complex, typically only the presence of thrombin is ascertained or detected.
  • the complex also can comprise thrombomodulin bound to the phospholipid vesicle.
  • methods of monitoring the effects of antithrombotic therapy comprising contacting a blood or plasma sample from the patient with a complex comprising tissue factor bound to a phospholipid vesicle, and determining the amount of active blood proteins in the sample, wherein the active blood proteins are at least one selected from the group consisting of activated protein C and thrombin.
  • thrombomodulin can be employed in order to determine the activation of protein C.
  • the complex also can comprise thrombomodulin bound to the phospholipid vesicle. Without the use or inclusion of thrombomodulin, typically only the level of thrombin is determined.
  • methods of assaying the level of activity in a preparation containing active blood proteins comprising contacting the preparation with a complex comprising tissue factor bound to a phospholipid vesicle, and determining the amount of active blood proteins in the sample, wherein the active blood proteins are at least one selected from the group consisting of activated protein C and thrombin.
  • the active blood proteins can be obtained from purified sources and comprise at least one protein selected from the group consisting of thrombin and activated protein C. These proteins can be obtained from natural sources, be recombinantly produced, and be made by peptide synthesis, in whole or in part.
  • the complexes employed can be like those described above, and can be optionally used in conjunction with thrombomodulin, as described above. In certain embodiments, the complex also can comprise thrombomodulin bound to the phospholipid vesicle.
  • FIGURE 1 panels A-H, depict thrombin activity of the PL-TF complexes identified in TABLE 1. Thrombin activity is measured in the presence of 27 nm TM (closed squares ⁇ ) or in the absence of TM (open squares D).
  • FIGURE 2 panel A depicts the initial rate of thrombin generation relative to TF concentration, and panel B depicts the TF surface density. This data is based upon the data of FIGURE 1.
  • FIGURE 3 depicts the effect of the initial rate of thrombin generation on maximum concentration of thrombin and APC, and on the feedback inhibition of thrombin generation.
  • thrombin activity is measured in the absence of TM.
  • panel B the maximum APC activity in the mean APC activity observed after 10 and 15 minutes of coagulation in the presence of TM.
  • Panel C depicts the percent of inhibition of thrombin generation , which was calculated from the peak thrombin activities detected in the presence and absence of TM.
  • FIGURE 4 depicts the rate of inactivation of purified human factor Va by APC, and shows that the inactivation rate depends on phospholipid vesicle concentration.
  • Panel A shows the time course of phospholipid vesicle concentration of 5 ⁇ m (closed circle, •), 20 ⁇ m (closed triangle, ⁇ ), 60 ⁇ m (closed square, ⁇ ) and 120 ⁇ m (closed, upside down triangle, T).
  • Panel B show the calculated pseudo first order rate constants as a function of the phospholipid content of the vesicles.
  • FIGURE 5 depicts the equivalent effect of TF surface density on the rate of APC- mediated factor Va inactivation and the feed inhibition of thrombin generation in plasma.
  • Pseudo first order rate constants if AP-mediated factor Va inactivation were measured using purified human proteins (panel A) and percent of thrombin generation inhibition in normal human plasma (panel B) in the presence of PL-TF complexes with low TF surface density (open squares D) or high TF surface density (closed squares, ⁇ ).
  • the present invention provides complexes of phospholipid vesicles and tissue factor (PL-TF complex).
  • the PL-TF complex can be used in the presence or absence of thrombomodulin (TM) in an assay system to measure prothrombotic and antithrombotic tendencies in a test sample, such as plasma from a patient.
  • thrombomodulin also can be bound to the phospholipid vesicle to form a complex comprised of a phospholipid vesicle, tissue factor and thrombomodulin.
  • the PL-TF complex (optionally with thrombomodulin accompanying the complex or as part of the complex itself by binding thrombomodulin to the phospholipid vesicle) thus serves as a reagent in this context, for example.
  • inventive subject matter is not limited to or defined by any type of theory or explanation. It is to be understood that the present inventions function by virtue of biological phenomenon and processes, and a complete understanding of the full nature of such biological phenomenon and processes are not necessary to successfully practice the inventive subject matter taught herein.
  • the PL-TF complex comprises phospholipid vesicles with tissue factor bound thereto (optionally including thrombomodulin bound to the vesicle).
  • bound in its various grammatical forms refers to an association between the tissue factor and the phospholipid vesicle that is of sufficient strength and integrity to permit the complex to be considered an entity that is not unduly disrupted or separated under physiologic conditions.
  • a single phospholipid vesicle may have 10 or more tissue factor molecules bound (i.e. , connected/attached) to the vesicle.
  • the phospholipid vesicles can be formed by a number of ways, such as: the extrusion method according to Olson et al, Biochem. Biophys. Ada 557: 9-23 (1979), reversed phase evaporation technique (F. Szoka and D. Paphadjopoulos, Proc. Nat'l Acad. Sci. USA 75: 4194-98 (1978)), sonication of phospholipid dispersions (Barenholz et al, Biochemistry 16: 2806-10 (1977); Gregoriades et al, FEBSLett. 14: 95-99 (1971)), the ethanol injection method (S. Batzri and E.D. Korn, Biochem. Biophys.
  • the PL-TF complex also can be used in conjunction with thrombomodulin (which optionally also can be bound to the phospholipid vesicle) for the generation of thrombin and activated protein C (APC), while dampening the effect of APC.
  • thrombomodulin which optionally also can be bound to the phospholipid vesicle
  • APC activated protein C
  • the PL-TF complex can be used to interfere with the APC-mediated inactivation of Factor Va and Factor Villa, thereby permitting the generation of further thrombin.
  • the PL-TF complex can be part of a preparation and an assay kit.
  • the PL-TF complex can be used in methods for evaluating the effects of antithrombotic compounds (for example, coumadin). Additionally, the complexes can be used to ascertain the levels/activities of blood proteins in the blood or plasma of a test subject or in a preparation containing blood or plasma.
  • Bovine serum albumin (BSA) was purchased from Calbiochem (La Jolla, CA). Viper venom Agkistrodon rhodostoma (Ancrod) and purified bovine thrombin were obtained from Sigma (St. Louis, MO). Chromogenic substrate S-2366 and 1-2581 thrombin inhibitor were obtained from Chromogenix (M ⁇ lndal, Sweden). Recombinant human tissue factor (TF) and recombinant soluble thrombomodulin (TM) were purchased from American Diagnostica, Inc. (Greenwich, CT).
  • DOPC l,2-dioleyl-sn-glycero-3-phosphocholine
  • POPS l-palmitoyl-2-oleyl-sn-glycero-3-phosphoserine
  • Prothrombin was purified from a human prothrombin complex fraction prepared according to Brummelhuis, METHODS OF PLASMA PROTEIN FRACTIONATION (Curling ed., Academic Press 1980) and subsequent purification to homogeneity as described in Turecek et al, Ann. Haemotol. 74 (Supp. II): All (1997).
  • Human factor Xa was prepared from highly purified factor X followed by activation with Russell's viper venom and affinity purification as described above. Turecek, loc. cit.
  • Defibrinated plasma samples were prepared as follows: Pooled normal plasma was a commercial product of George King Bio-Medical, Inc. (Overland Park, KS). The frozen samples were thawed at 37°C, defibrinated with Ancrod (Sultan et al, J. Lab. Clin. Med. 121: 444 (1993)), aliquoted and frozen at -80°C, then thawed at 37°C just before use.
  • Phospholipid vesicle - tissue factor (PL-TF) complex was done in the following way: Phospholipid vesicles composed of 80% (w/w) of DOPC and 20% (w/w) of POPS were prepared by the extrusion method (Hope et al., Biochim. Biophys. Acta. 812: 55 (1985)) as follows: A phospholipid thin film was hydrated with 20 mM Tris, 150 mM NaCl, pH 7.4 (TBS) freeze-dried and reconstituted with the appropriate volume of distilled water.
  • TBS pH 7.4
  • the phospholipid suspension was repeatedly extruded through two stacked 1000 nm polycarbonate filters, then through two 400 nm polycarbonate filters using an extrusion device of Lipex Biomembranes, Inc. (Vancouver, Canada).
  • the vesicle preparation was diluted with TBS to a concentration of 1 .2 mM and freeze-dried after addition of 5 % sucrose (w/v). After reconstitution, the vesicles had a mean diameter of about 260 nm (polydispersity 0.23), as determined by dynamic light scattering (Zetasizer 4, Mai vern Instruments, Worcestershire, UK) (Ruf et al., Meth. Enzymol. 172: 364-90 (1989).
  • Electron microscopic studies with negative staining or thin section preparations showed that about ninety percent of the vesicles in the preparation are unilamellar.
  • Recombinant TF non-lipidated, American Diagnostica, Inc., Greenwich, CT
  • was added to the reconstituted vesicle preparation frozen at -20 °C overnight, thawed for 30 minutes at 25 °C and diluted 6.7-fold with TBS to yield final concentrations of 0.28 to 105 nM TF and 120 ⁇ M PL.
  • thrombomodulin also can be included in this and other production methodologies to yield complexes comprising phospholipid vesicles with tissue factor and thrombomodulin bound thereto.
  • TF that is, a PL-TF complex
  • Vesicle size decreased upon storage at 4°C for several days to about 200 to about 300 nm, which is similar to the size of vesicles present in the supernatants, suggesting that the aggregates dissociate to monomeric forms.
  • TF concentration of pellets and supernatants was measured with the IMUBIND ELISA system (American Diagnostica, Inc., Greenwich, CT) and the phospholipid concentration was calculated on the basis of the phosphatidylcholine content using an enzymatic assay (Test Combination Phospholipids, Boehringer Mannheim, Germany) for all preparations. No difference was observed in the TF content of the vesicles measured in the absence or presence of detergents, suggesting that all TF bound onto the surface of the PL vesicles, and not inside. With increasing amounts of TF, phospholipids were distributed increasingly in the supernatants, suggesting that PL-TF complexes with high TF concentration are less likely to aggregate to form a pellet.
  • TF surface density was calculated as TF molecules per vesicle, assuming that all phospholipids are arranged as unilamellar spheres of an average diameter of about 260 nm and that TF is fully bound to phospholipid vesicles (Pitlick et al., Biochemistry 9: 5105- 13 (1970)) and that TF is evenly distributed on a lipid bilayer composed of 5 x 10 6 molecules/ ⁇ m 2 . See Alberts et al, MOLECULAR BIOLOGY OF THE CELL (Garland Publishing, Inc. 1989).
  • the TF surface density was comparable in the corresponding pellets and supernatants derived from the same preparations.
  • the PL-TF complexes are divided into low and high TF surface density groups, as shown in Table 1 below.
  • a volume of 100 ⁇ l of defibrinated plasma was preincubated with 50 ⁇ l of PL-TF complex "mix” or "pellet” in the presence of absence of 50 ⁇ l TM (270 nM) in 25 mM HEPES, 175 mM NaCl, 1 mg/ml BSA, pH 7.35 (HNaBSA) buffer (final volume: 450 ⁇ l) at 37°C for 5 minutes.
  • the coagulation pathway was initiated with 50 ⁇ l of 25 mM CaCl 2 and subsamples were withdrawn after pre-specified incubation intervals at 37°C.
  • Thrombin activity was measured in 10 ⁇ l subsamples that were added to 300 ⁇ l TH-1 chromogenic substrate containing 3 mM EDTA to stop further thrombin generation.
  • chromogenic substrate conversion was terminated with 100 ⁇ l of acetic acid (75% v/v).
  • APC generation was measured in 30 ⁇ l subsamples, added to 300 ⁇ l S-2366 chromogenic substrate containing 3 mM EDTA and 0.1 mM 1-2581 thrombin inhibitor to block thrombin activity.
  • 100 ⁇ l of acetic acid (75% v/v) was added to terminate the chromogenic substrate conversion.
  • 300 ⁇ l reaction mixture was transferred to an ELISA plate, and the absorbance was measured at 405 nm in an ELISA-reader.
  • Purified factor Va (0.375 nM) was incubated with 0.05 mM APC in the presence of phospholipid vesicles alone or PL-TF complex and 5 mM CaCl 2 in HNaBSA buffer at 37°C.
  • Factor Va activity was measured in a prothrombinase assay as follows: Subsamples (10 ⁇ l) were removed at pre-specified time intervals, added to a 140 ⁇ l mixture of 0.05 nM factor Xa, 75 nM of prothrombin, 10 ⁇ M PL (PC:PS vesicles, 80:20) and 5 mM CaCl 2 in HNaBSA buffer.
  • thrombin activity was measured in 10 ⁇ l subsamples as noted above using TH-1 chromogenic substrate. Without the addition of APC, factor Va was stable for at least 10 minutes at 37°C, and the activity measured was taken at 100% .
  • reaction conditions of the prothrombinase assay were chosen such (low factor Xa concentration) that APC-mediated cleavage at Arg 506 already results in a complete loss of factor Va activity and that slow cleavage at Arg 306 negligibly contributes to factor Va inactivation.
  • thrombin generation was induced with PL-TF complexes that had high TF concentrations (1.3-32 nM) and high TF surface densities (78-306 TF molecules/phospholipid vesicle), the rate of thrombin generation and peak thrombin activities were higher. Moreover, no difference was found when TM was present.
  • the maximum thrombin generated in the absence of TM was a function of the initial rate of thrombin generation (FIGURE 3, Panel A).
  • APC generation started simultaneously with thrombin activation, reaching plateau levels within 10 to 15 minutes (data not shown). No APC generation was detected in the absence of TM.
  • both the rate of activation (not shown) and the maximum of APC generated was in direct correlation with the initial rate of thrombin generation (FIGURE 3, Panel B). Inhibition of thrombin generation, calculated from the maximum thrombin concentration obtained with and without TM, would be expected to be highest in the presence of the highest APC concentration.
  • TF pathway-induced thrombin generation is mainly regulated by the rate of factor Va activation via a thrombin feedback effect and by the rate of factor Va inactivation by APC.
  • Such inactivation requires the presence of TM, which complexes with generated thrombin and activates protein C in plasma.
  • TM complexes with generated thrombin and activates protein C in plasma.
  • the PL-TF complexes were assessed for factor Va inactivation by APC using purified proteins in a non-plasma milieu.
  • phospholipid concentration is a critical determinant for factor Va inactivation (FIGURE 4) indicating that the phospholipid vesicle surface serves as a binding platform for interaction of the reaction components.
  • the time course of APC-mediated factor Va inactivation was measured in the presence of constant amounts of purified human factor Va and APC and increasing concentrations of phospholipid vesicles without TF (FIGURE 4, Panel A).
  • the initial rate of inactivation was directly related to phospholipid concentration and the calculated pseudo first order rate constants (k') of factor Va inactivation appear to be a function of the concentration of vesicles (FIGURE 4, Panel B).
  • Factor Va inactivation measured in the presence of phospholipid preparations with low TF surface density showed comparable inactivation rates as calculated for the corresponding phospholipid vesicles without TF.
  • the factor Va inactivation rate constants measured in the presence of high TF surface density phospholipid vesicles were markedly lower than obtained for phospholipid vesicles without TF (TABLE 2).
  • Rate constants were estimated from the reference curve shown in Fig. 4, Panel B.
  • the rate of purified factor Va by APC in the non-plasma system showed the same relationship to TF surface density (FIGURE 5, Panel A) as did the degree of thrombin generation inhibition in the plasma system (FIGURE 5, Panel B).
  • This comparison of the same PL-TF complexes in the two reaction systems indicates that TF surface density controls feedback inhibition of thrombin generation by limiting APC-mediated factor Va inactivation.
  • the rate of factor Va inactivation was strongly influenced by the TF surface density on the vesicles.
  • the apparent first order rate constants of APC- mediated factor Va inactivation were comparable with those determined on phospholipid vesicles without TF.
  • TF surface density exceeded a threshold of more than 10 TF molecules per vesicle, the rate of factor Va inactivation was greatly reduced and became independent of the phospholipid concentration.
  • the rate of factor Va inactivation by APC is inversely related to the tissue factor surface density (molecules/vesicle) of the complexes, providing an explanation for the "feedback inhibition" effect.
  • TF act in concert to promote clot formation (hemostasis) or, in pathologic states, to enhance thrombus formation.
  • TF induces maximal early thrombin generation.
  • APC also is generated maximally, the high TF surface density would limit available vesicle surface for APC-mediated factor Va inactivation. Both processes are conducive to effective physiologic hemostatic plug formation. Under conditions of low TF presence, initial thrombin generation rates are low and APC-mediated factor Va inactivation has ample phospholipid vesicle surface for rapid action, effects which would limit thrombin generation and fibrin formation.
  • TF modulates both thrombin formation and factor Va inactivation, upregulating the former while downregulating the latter for maximal hemostasis or setting imits on the former while allowing the latter in situations of natural inhibition of clot formation by the protein C pathway.
  • EXAMPLE 9 Reagents and assays 1.
  • the PL-TF complex should be employed with a defined TF concentration and surface density.
  • the vesicle is unilamellar and has a diameter of 100 to 500 nm, preferably 200 nm to 300 nm, although vesicles outside these ranges also can be readily employed by the skilled person in view of the teachings contained herein. , and thus are within the scope of the invention.
  • the phospholipid vesicles can be made with a variety of phospholipids, such as l,2-dioleyl-sn-glycero-3-phosphocholine (DOPC) and l-palmitoyl-2- oleyl-sn-glycero-3-phosphoserine (POPS).
  • DOPC l,2-dioleyl-sn-glycero-3-phosphocholine
  • POPS l-palmitoyl-2- oleyl-sn-glycero-3-phosphoserine
  • DOPC l,2-dioleyl-sn-glycero-3-phosphocholine
  • POPS l-palmitoyl-2- oleyl-sn-glycero-3-phosphoserine
  • One preferred embodiment employs vesicles comprised of 80% (w/w) DOPC and 20% (w/w) POPS.
  • the presence of bound tissue factor should be sufficiently large (at least about 10 TF molecules per vesicle) so as to permit the capabilities of the invention to be best realized.
  • the PL-TF complex can be part of an assay kit, which can include, among other things, thrombomodulin.
  • assays kits are useful for the simultaneous thrombin and activated protein C (APC) generation.
  • APC activated protein C
  • thrombomodulin TM
  • thrombin is generated by calcium initiation of the coagulation pathways, following which the generated thrombin initiates PC activation and APC accumulation.
  • the complexes also can comprise bound thrombomodulin such that the vesicle has tissue factor and thrombomodulin bound thereto.
  • the assay kits including the PL-TF complex, also are useful for factor Va inactivation.
  • APC-mediated factor Va inactivation also can be measured in purified systems using PL-TF complexes for the surface-oriented reaction.
  • PL-TF complexes can be used in a variety of contexts. For example, these complexes can be used to simultaneously measure and evaluate prothrombotic and antithrombotic tendencies. The following are illustrative (and non-limiting) of the uses for PL-TF complexes:
  • heparin anti-thrombin or new anti-thrombin agents fail to evaluate intrinsic APC generation, the balance of which is important in effective prophylaxis and prevention of thrombotic complications.
  • Novel anticoagulants can be studied by such a functional approach.
  • the PL-TF complex is an ideal analytical tool for studies of genetics of coagulation, evaluation of recombinant mutants and protein mimetics of blood factors (for example, prothrombin, protein C, etc.), classical clotting biochemistry, drug or antibody influences, and development of new anti-thrombotic and hemostatic approaches.
  • blood factors for example, prothrombin, protein C, etc.
  • protein mimetics of blood factors for example, prothrombin, protein C, etc.
  • classical clotting biochemistry for example, drug or antibody influences
  • development of new anti-thrombotic and hemostatic approaches for studies of genetics of coagulation, evaluation of recombinant mutants and protein mimetics of blood factors (for example, prothrombin, protein C, etc.), classical clotting biochemistry, drug or antibody influences, and development of new anti-thrombotic and hemostatic approaches.

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Abstract

L'invention concerne des complexes constitués de thromboplastine tissulaire liée à une vésicule phospholipidique. Ladite vésicule phospholipidique peut comprendre de la 1-2-dioleyl-sn-glycéro-3-phosphocholine (DOPC) et de la 1-palmitoyl-2-oléyl-sn-glycéro-3-phosphosérine (POPS) et présenter un diamètre, par exemple, d'environ 200 nm à 300 nm. Les vésicules peuvent être monolamellaires, et le complexe peut comprendre plusieurs molécules de thromboplastine tissulaire (de préférence 10 ou plus) liées à la vésicule phospholipidique. Eventuellement, de la thrombomoduline peut être liée aux vésicules. Des procédés d'utilisation desdits complexes et de préparations comprenant ces complexes sont également décrits.
PCT/US1999/014183 1998-06-23 1999-06-23 Preparations de complexes thromboplastine tissulaire-vesicule phospholipidique, leurs procedes de fabrication et d'utilisation WO1999066939A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU47093/99A AU4709399A (en) 1998-06-23 1999-06-23 Phospholipid vesicle-tissue factor complex preparations and methods of making and using same

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US9041798P 1998-06-23 1998-06-23
US60/090,417 1998-06-23

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WO1999066939A1 true WO1999066939A1 (fr) 1999-12-29

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
WO2006089324A1 (fr) * 2005-02-22 2006-08-31 Technoclone Gesellschaft M.B.H. Procede de determination de l'activation de coagulation et dispositif destine a la mise en oeuvre du procede

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Publication number Priority date Publication date Assignee Title
US5051357A (en) * 1989-07-14 1991-09-24 Board Of Trustees Operating Michigan State University Method and assay using inactivation of factors Va and VIIIa by activated Protein C to diagnose thrombic disease or assay for Protein C and kit therefor
EP0680763A2 (fr) * 1994-05-06 1995-11-08 Immuno Ag Préparation stable pour le traitement des anomalies de la coagulation du sang comprenant un facteur de coagulation activé et des vésicules lipidiques
US5643739A (en) * 1993-12-03 1997-07-01 Immuno Ag Assay for determining sensitivity to activated protein C
US5698677A (en) * 1994-05-06 1997-12-16 Immuno Aktiengesellschaft Stable preparation for the treatment of blood coagulation disorders

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5051357A (en) * 1989-07-14 1991-09-24 Board Of Trustees Operating Michigan State University Method and assay using inactivation of factors Va and VIIIa by activated Protein C to diagnose thrombic disease or assay for Protein C and kit therefor
US5643739A (en) * 1993-12-03 1997-07-01 Immuno Ag Assay for determining sensitivity to activated protein C
EP0680763A2 (fr) * 1994-05-06 1995-11-08 Immuno Ag Préparation stable pour le traitement des anomalies de la coagulation du sang comprenant un facteur de coagulation activé et des vésicules lipidiques
US5698677A (en) * 1994-05-06 1997-12-16 Immuno Aktiengesellschaft Stable preparation for the treatment of blood coagulation disorders

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Title
ARKEL Y S, ET AL.: "A HIGHLY SPECIFIC FUNCTIONAL TEST FOR FACTOR V LEIDEN: A MODIFIED TISSUE FACTOR ASSAY FOR ACTIVATED PROTEIN C RESISTANCE", HAEMOSTASIS, BASEL,, CH, vol. 27, 1 June 1998 (1998-06-01), CH, pages 290 - 304, XP002924058, ISSN: 0301-0147 *
DATABASE CAPLUS ON STN, AN 1999:8211 POTZSCH et al., "Method and Test Kit for Diagnosis of Dysprothrombinemia by Determining Anticoagulant Thrombin in Blood or PLasma"; & WO 9857178 A1, 17 December 1998. *
DUCHEMIN J, ET AL.: "A NEW ASSAY BASED ON THROMBIN GENERATION INHIBITION TO DETECT BOTH PROTEIN C AND PROTEIN S DEFICIENCIES IN PLASMA", THROMBOSIS AND HAEMOSTASIS, SCHATTAUER GMBH, DE, vol. 71, no. 03, 1 March 1994 (1994-03-01), DE, pages 331 - 338, XP002924059, ISSN: 0340-6245 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006089324A1 (fr) * 2005-02-22 2006-08-31 Technoclone Gesellschaft M.B.H. Procede de determination de l'activation de coagulation et dispositif destine a la mise en oeuvre du procede
US7767458B2 (en) 2005-02-22 2010-08-03 Technoclone Gesellschaft M.B.H. Method for determining coagulation activation and device for carrying out said method

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