text:
|Publication number
|Publication type
|Application number
|Publication date
|Jun 11, 1996
|Filing date
|Jun 7, 1995
|Priority date
|Oct 4, 1991
|Fee status
|Also published as
|Publication number
|John T. Matschiner
|Original Assignee
|Matschiner; John T.
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (13), Non-Patent Citations (2), Referenced by (32), Classifications (13), Legal Events (7)
|External Links: USPTO, USPTO Assignment, Espacenet
This application is a continuation of application Ser. No. 07/771,644, filed Oct. 4, 1991, now abandoned.
The present invention relates to a novel one-stage blood coagulation assay which incorporates soluble thrombomodulin for the rapid and direct in vitro determination of the functional status of vitamin K-dependent coagulation-inhibiting proteins in a plasma sample. In particular, the functional status of protein C and protein S are reliably and sensitively determined with this assay, without separation from the plasma sample in which they are contained. In addition, the present invention relates to novel molecular complexes produced in the aforementioned one-stage blood coagulation assay.
The capacity of blood to clot and stop flowing from a wound is dependent on the proper functional interaction of a large number of factors and cofactors in the blood coagulation cascade. The ability of clinical laboratories to reliably and conveniently assay for these factors and cofactors in plasma samples from donor patients can be critical in monitoring individuals for whom either inappropriate coagulation episodes (such as occurs, for example, in disseminated intravascular coagulation disease) or inappropriate failure of blood to clot (such as occurs, for example, in hemophilia) is a daily life-threatening problem.
The physiologic basis for these clinically important coagulation problems can be exceedingly complex to diagnose. In most cases, however, diagnosis can be made by detecting either quantitative or functional changes in certain of the biological factors and cofactors which activate or regulate the coagulation process. A number of diagnostic assays have been developed which reliably measure many of the regulatory proteins involved in the coagulation cascade (see, for example, the textbooks by R. Hoffman et al., Hematology: Basic Principles and Practice, New York: Churchill Livingston Inc., 1991; and by C. Kjeldsberg et al., Practical Diagnosis of Hematologic Disorders, Chicago: American Society of Clinical Pathologists Press, 1989). These include such routine coagulation assays as the activated partial thromboplastin time (APTT) assay, the prothrombin time (PT) assay, and the thrombin clotting time (TCT) assay. In spite of extensive research, none of these assays directly measure, in a one-stage assay, the functional status in plasma of the vitamin K-dependent coagulation-inhibiting proteins, protein C and protein S. Because protein C and protein S are both essential in the normal regulatory process of down-regulating the blood coagulation cascade, the lack of any one-stage assay which directly determines both of their functional activities remains a serious deficiency in the art.
The need for a convenient one-stage assay for determining protein C and protein S function in plasma is emphasized by the fact that abnormalities in protein C and protein S function are more common in the population than is usually believed. For example, from 4-to-5% of the population as a whole have genetically-acquired protein C or protein S deficiencies, and a large number of these individuals have a clinical history of recurrent thrombotic (microvascular clotting) episodes (Gladson et al., Thromb. Haemostas. 59: 18, 1988). It is these individuals who should be regularly monitored for the functional activities of these coagulation-inhibiting proteins individuals who are known to be at high risk for recurrent thrombosis (which can be lethal) should be kept on lifelong maintenance anticoagulant therapy, with regular monitoring.
The coagulation enzyme system
To better appreciate the importance of such assays, and how activated protein C and protein S are involved in regulating the blood coagulation cascade, the following brief description of the coagulation enzyme system is provided.
The blood clotting system may best be viewed as a chain reaction involving the sequential activation of inactive enzyme precursors (zymogens) into active serine proteases. These activation events, which take place on the surfaces of cells such as platelets, white blood cells, and endothelial cells, have been divided into two distinct pathways termed the extrinsic and the intrinsic pathways of coagulation. A clot is generated in the intrinsic pathway by activation of those coagulation components which are all contained in (or are "intrinsic to") whole blood. In the extrinsic pathway, components intrinsic to whole blood are reguired along with an externally-supplied coagulation-activating substance known as "tissue factor" (also sometimes referred to as thromboplastin, thrombokinase, or blood coagulation factor III). Tissue factor is a cell surface protein extrinsic to blood, and is expressed by cellular injury. Whether a particular coagulation factor becomes activated in the extrinsic or the intrinsic pathway is important in selecting a particular coagulometric assay with which to detect and evaluate that particular factor.
Thrombin. The multistep coagulation chain reaction uitimately produces the enzyme thrombin, the last serine protease in the coagulation cascade, which, through limited proteolysis, converts fibrinogen molecules into an insoluble gel of fibrin fibers which forms the physical clot. Two key events in the coagulation cascade are the conversion of clotting factor X into an activated form, factor Xa, and the subsequent conversion of prothrombin by factor Xa into thrombin. Both of these conversion reactions occur on cell surfaces, such as, for example, the surfaces of platelets, and both reactions require cofactors. These cofactors, factors V and VIII, are in circulation in the form of relatively inactive precursor molecules. When the first few molecules of thrombin are formed, thrombin loops back and activates factors V and VIII through limited proteolysis. The activated factors, Va and VIIIa, accelerate both the conversion of prothrombin into thrombin and also the conversion of factor X to factor Xa, speeding the clotting process by approximately 100,000 fold. This significant amplification is essential in the timely formation of a clot. As will be discussed below, factors Va and VIIIa are the principal protein targets of activated protein C and protein S.
Thrombomodulin. Some of the thrombin formed during the formation of a clot is also bound by thrombomodulin, an essential reagent used in soluble form in the present invention. Thrombomodulin is a glycoprotein normally found fixed in the surface membranes of endothelial cells that form the lining of all blood vessels. While vascular endothelium once was thought to be a passive barrier which simply channeled the blood, it now is known that endothelial cells are actively involved in the regulation of intravascular coagulation mechanisms, and that much of this activity is due to the thrombomodulin in the endothelial cell membranes.
Thrombin-thrombomodulin complex. Thrombomodulin forms a tight, stoichiometric complex with thrombin, altering both the physical shape and functional properties of thrombin so that it no longer has procoagulant activity, i.e., it no longer converts fibrinogen to fibrin, activates platelets, or converts clotting factors V and VIII to their activated counterparts Va and VIIIa. Rather, the thrombin which is bound by thrombomodulin becomes an efficient activator of protein C. The rate constant for the activation of protein C by thrombin bound in a thrombomodulin-thrombin complex is about 20,000 fold higher than the rate constant for activation of protein C by thrombin found free in solution. Additionally, activation of protein C by thrombin in the absence of thrombomodulin is inhibited by calcium ions, whereas activation by the thrombin/thrombomodulin complex is a calcium-dependent reaction.
Thus, the coagulation-inhibiting effects of thrombomodulin are of two different types. One is a heparin-like anticoagulant effect in which the binding of thrombin to thrombomodulin inhibits the capacity of thrombin to enzymatically convert fibrinogen into fibrin fibers; the anticoagulant heparin exerts a similar antithrombin effect. This is the thrombomodulin-mediated anticoagulant effect most commonly observed by others (for example, see Gomi et al., Blood 75: 1396, 1990).
The second anticoagulant effect of thrombomodulin results from the activation of protein C by the thrombin-thrombomodulin complex. This effect is the key component of the present invention. When activated, protein C has the capacity to enzymatically cleave (via hydrolysis) both factor Va and factor VIIIa, substantially inhibiting both of their clot-promoting activities. In the presence of its vitamin K-dependent cofactor protein S, the rate of activated protein C-mediated hydrolysis of factors Va and VIIIa is increased by about 25 fold compared to hydrolysis in the absence of protein S. Thus, one of the important mechanisms operating in the vascular endothelium to maintain the normal anticoagulant state of the endothelial surface is the pathway by which a thrombin-thrombomodulin complex activates protein C to form a coagulation-inhibiting activated protein C-protein S complex on a cell surface.
The vitamin K-dependency of the protein C and protein S coagulation-inhibiting proteins derives from the fact that a vitamin K-dependent microsomal carboxylase enzyme in the liver forms an unusual amino acid, gammacarboxyglutamic acid, in a post-ribosomal carboxylation step in the precursor proteins of both protein C and protein S. The appearance of the gammacarboxyglutamic acid moieties in these protein molecules is crucial in that it facilitates their efficient binding via a calcium-mediated bridge, to phospholipid-containing surfaces such as, for example, the surfaces of platelets, endothelial cells, and, importantly for in vitro coagulometric assays, phospholipid micelles in solution. As noted below, the binding of the carboxylated glycoproteins to a phospholipid surface allows the proteins to concentrate, interface and interact with one another more efficiently in three-dimensional space. Several in vitro clotting assays take advantage of this fact, in that phospholipids are included among the necessary reagents used in the coagulometric assays.
Phospholipid. All clotting reactions with meaningful rates are viewed as occurring on a surface. Crucial to the efficient interaction of thrombomodulin (the cofactor), thrombin (the enzyme), and protein C (the substrate) is the presence of calcium ions and an integrating surface such as is found, for example, on a soluble phospholipid micelle (a submicroscopic phospholipid sphere). On such a surfacer the three reactants are brought into close proximity, thereby substantially increasing their effective concentrations and their reaction rate many fold. It is believed that all relevant soluble complexes which contain soluble thrombomodulin, thrombin and activated protein C, and which produce vitamin K-dependent anticoagulant activity, also contain an essential surface component. The in vitro clotting assays of the present invention take advantage of this fact by the use of a suitable phospholipid (cephalin) in a preferred embodiment.
Protein C and Protein S assays
Until the present invention, it has not been possible to conveniently measure the functional activities of both protein C and protein S in a simple and rapid one-stage in vitro assay. This is because soluble thrombomodulin has never before been added directly into laboratory coagulometric assays to detect protein C zymogen. Rather, thrombomodulin has been added with thrombin as a preactivation reagent, either as a soluble complex, or in an immobilized form and in conjunction with a process which required either prior separation of protein C from other plasma components or the subsequent removal of activated protein C from other plasma components in order to carry out the final determination of activated protein C in a separate coagulometric or amidolytic assay. Thus, the anticoagulatory effect of soluble thrombomodulin, in the form of soluble thrombomodulin-thrombin complexes, has never been exploited directly in conventional clinical assays.
Protein C assays
For protein C, a number of different assays are available for its determination (as reviewed in detail by Lobermann et al., Behring Inst. Mitt. 79:112, 1986; Vigano-D'Angelo et al., in Biotechnology in Clinical Medicine [Albertini et al., editors] New York: Raven Press, 1989: and more recently by Preissner, Clin. Sci. 78: 351, 1990). The assays generally fall into three categories: antigenic detection assays, chromogenic (amidolytic) assays, and functional coagulometric assays.
Protein C antigenic assays. For determination of protein C antigen concentration in plasma, detection assays include electroimmunoassay, radioimmunoassay, and ELISA-type assays. Not only polyclonal antibodies from rabbit or goat, but also monoclonal antibodies have been used as detecting reagents in these assays. While in vitro detection of protein C with antibodies is a sensitive procedure, antigenic detection provides little or no information regarding the functional capacity of the protein C detected.
Protein C chromogenic assays. Alternatively, because activated protein C is an enzyme, its presence in a plasma sample can be quantified with a chromogenic substrate. Prior activation of protein C, with exposure of the enzymatic active site, is required for expression of its enzymatic activity. However, the relatively small size of the synthetic chromogenic substrate (about 600 daltons) commonly used in these assays may permit satisfactory proteolysis of the synthetic substrate, while missing defects in the functional integrity of protein C which would prevent proteolytic cleavage of factor Va and VIIIa, the biological substrates of activated protein C which each have molecular weights of about 300,000 daltons. Moreover, the interaction of activated protein C with its cofactors (protein S, calcium ions, and phospholipid), which requires that all functional features of the protein C molecule be intact, is not evaluated by chromogenic assays. It is important to be aware of this, since protein C molecules detected in chromogenic assays can still be deficient in functional anticoagulant capacity.
Protein C coagulometric assays. For determination of functional capacity of protein C molecules in a plasma sample, coagulometric assays offer the advantage of evaluating the coagulation-inhibiting activity of activated protein C in the presence of its usual biological substrates and cofactors, thereby reflecting more accurately the physiologic state of the protein C in the particular plasma sample being evaluated.
The protein C in a plasma sample is commonly activated before it is assayed. This activation is done either with or without prior separation of protein C from other plasma components. Isolation of the protein C from the test plasma sample has (up until the present invention) been necessary if the protein C is to be activated by thrombin or by the physiological activator, which is the thrombin/thrombomodulin complex. Unfortunately, because of poor reaction kinetics, activation of protein C with thrombin in the absence of thrombomodulin does not lead to complete activation of protein C in the sample. In contrast, when thrombin is bound to thrombomodulin, the rate of activation is increased by about 20,000 fold over the rate obtained with thrombin alone.
Activation can also be carried out without prior isolation of protein C from the test plasma sample by use of a snake venom activator such as, for example, southern copperhead venom, or the "PROTAC� C" reagent (the latter from Pentapharm, Basel, Switzerland).
After either of the above procedures, the anticoagulant activity of activated protein C is assessed most commonly either by prolongation of the otherwise routine activated partial thromboplastin time (APTT) assay, or in a factor Xa one-stage coagulometric assay. The clot time in this latter assay is a function of the conversion of prothrombin to thrombin, and is initiated by the addition of exogenous factor Xa to the plasma sample in the presence of calcium ions and a phospholipid component. The assay is sensitive to activated factor V, but not to activated factor VIII. In this system, addition of preactivated protein C from the test plasma sample to a control plasma sample prior to the addition of factor Xa prolongs the clot time as a result of its ability to inactivate factor Va generated during the reaction.
In stark contrast to the commonly used assays discussed above, the novel assay of the present invention does not require preliminary isolation of protein C from a plasma test sample; it permits the activation of protein C with its physiological activator, the thrombin/thrombomodulin complex; and it results in a determination of protein C functional activity in the test sample, all in a simple one-stage procedure.
Protein S assays
Laboratory evaluation of protein S status is complicated by the fact that two forms of protein S are present in plasma. In plasma, about 40% to 50% of the protein S is free and serves as the cofactor for activated protein C. The remaining 50% to 60% of plasma protein S is complexed to C4b binding protein ("C4bBP") and, as such, is unavailable as an anticoagulant. That C4bBP is an acute phase protein and is elevated during inflammation further complicates evaluation of protein S status. This rise in C4bBP favors a transient shift of the protein S to the complexed form, thereby inducing a relative and transient protein S deficiency state. Because protein S which is bound to C4bBP in plasma is no longer able to function as a cofactor with activated protein C, individuals with serious inflammatory disorders are often at risk for thrombosis. This is an acquired situation which will rise and fall, and even disappear, depending on the state of the inflammatory disorder.
In the clinical laboratory, a very limited number of tests are available to assess protein S status, and these are generally antigen detection assays. These tests include polyethylene glycol precipitation of bound protein S with subsequent measurement of the free protein S remaining in the plasma; crossed immunoelectrophoresis for protein S, which separates free and bound forms of protein S but is not quantitative; and enzyme-linked immunosorbent assays (ELISA). ELISA assays are suitable in combination with PEG precipitation to quantitate free protein S. However, while ELISA assays may detect protein S with sensitivity, such antigen detecting assays provide little or no information regarding the functional capacity of the protein S detected. Unfortunately, standardized functional assays for protein S are not yet available in the general clinical laboratory.
It is, therefore, a principal object of the present invention to provide a convenient and reliable one-stage assay by which both of the vitamin K-dependent coagulation-inhibiting proteins, protein C and protein S, can be quantitatively and functionally determined in a blood plasma test sample in a clinical laboratory.
It is another object of the present invention to provide a soluble coagulation-inhibiting complex comprising soluble thrombomodulin, thrombin and protein C, bound to a soluble phospholipid surface. Such a phospholipid surface is found, for example, on soluble phospholipid micelles.
It is still another object of the present invention to provide a soluble complex comprising activated protein C and its cofactor, protein S, bound to a soluble phospholipid surface.
Yet another object is to provide a soluble complex comprising activated protein C, protein S and factor Va bound to a soluble phospholipid surface.
Still another object is to provide a soluble complex comprising activated protein C, protein S and factor VIIIa bound to a soluble phospholipid surface.
In accordance with the present invention, there is provided a novel one-stage assay which uses soluble thrombomodulin for determining in a novel blood coagulation assay the functional status of vitamin K-dependent coagulation-inhibiting proteins in plasma. The method comprises combining a blood plasma sample and a coagulation-activating substance in a calcium-free solution, then adding soluble thrombomodulin and calcium ions in amounts sufficient to initiate a coagulation reaction, measuring the time required for the plasma test sample to clot, comparing the measured clot time to that of standard control plasma samples, and then using the compared clot time values for determining the risk of thrombosis in the individual from whom the plasma test sample was taken.
In a preferred embodiment of the present invention, soluble thrombomodulin is added simultaneously with the calcium ions (i.e., the recalcification step), and the subsequent extension of the clot time caused by the anticoagulant effect of the soluble thrombomodulin is compared to the extension of clot time that is observed in protein C- and/or protein S-deficient plasma samples used as deficient plasma controls.
The vitamin K-dependent coagulation inhibiting factors with which the present invention is principally concerned are protein C and protein S, both of which are essential regulatory factors in maintaining the anticoagulatory state of the endothelial cell lining of all blood vessels in the body. Any other coagulation-inhibiting protein, the functional status of which may be determined in these assays by using the methods taught herein, are also included within the scope of the present invention.
The coagulation-activating substance added to the mix of reagents at the beginning of the assay of this invention is, in the preferred embodiment, the "DADE�/ACTIN� ACTIVATED CEPHALOPLASTIN REAGENT". This reagent is available commercially from the Baxter Healthcare Corporation and is more fully described in its package insert No. L10293-BH-H (revised 10/88) (Baxter/Dade, Miami, Fla.), the content of which is incorporated herein by reference. The coagulation-activating ingredient in this reagent is ellagic acid, a negatively-charged organic compound of formula C14 H6 O8, having a molecular weight of about 302 daltons.
In another embodiment, the coagulation-activating substance is factor Xa. Factor Xa acts upon prothrombin in the presence of factor V, calcium ions and phospholipid to form a complex which activates prothrombin, converting it to thrombin.
In another embodiment, the coagulation-activating substance is a reptilase, preferably, Russell's viper venom, which is available from a variety of commercial sources. Russell's viper venom is an activator of factor X, enzymatically cleaving factor X to generate factor Xa, which is the procoagulant factor just discussed.
In yet another embodiment, the coagulation activating substance is a protein known as "tissue factor", which is a cell surface protein released into blood following cellular damage. Tissue factor is a membrane receptor for factor VII, and is available from a variety of commercial sources. In conjunction with tissue factor and calcium ions, factor VII enzymatically modifies factor X, converting factor X to its procoagulant form, factor Xa, discussed above.
In still other embodiments, coagulation activating substances include negatively-charged collagen molecules; or negatively-charged foreign surfaces such as micronized silica, kaolin, fine glass or metal beads, and even wooden applicator sticks. The use of these latter particulates are limited, however, to assays in which the presence of such insoluble foreign surfaces will not interfere in carrying out the assay.
In the preferred embodiment of the present invention, the phospholipid added to the assay is cephalin, a glycerophosphoric acid extracted from dehydrated rabbit brain, and contained as an active ingredient in the "DADE�/ACTIN� ACTIVATED CEPHALOPLASTIN REAGENT", as more fully described in the Baxter Healthcare Corporation package insert No. L10293-BH-H [revised 10/88] (Baxter/Dade, Miami, Fla.). Biochemically, cephalins consist of glycerophosphoric acid in which the two free hydroxyls of the molecule are esterified with long-chain fatty acid residues, and ethanolamine forms an ester linkage with the phosphate group. All natural cephalin products occur in alpha-forms, which have the following generic structure: ##STR1## where R and R' can be, but are not necessarily, identical fatty acids.
Other phospholipids which may be used in further embodiments of the present invention are those which successfully facilitate the activation of protein C and its detection in the coagulometric assays of the present invention. It is believed that all relevant soluble complexes which contain soluble thrombomodulin, thrombin and activated protein C, and which produce vitamin K-dependent anticoagulant activity, also contain an essential surface component. The in vitro clotting assays of the present invention take advantage of this fact by the use of a suitable phospholipid (cephalin) in a preferred embodiment. However, besides phospholipids, any micelle-forming (or liposome-forming) ingredient which is amphipathic and whose charge distribution and size make it optimal for use as a surface carrier in facilitating the coagulometric assays disclosed in the present invention is included within the scope of the coagulation-activating agents of the present invention.
By the term "normal control plasma" as used above and hereinafter is meant a plasma sample which contains the usual (statistically common) and expected amounts of all known coagulation-related factors which are essential in promoting the clotting of plasma by either the intrinsic or the extrinsic coagulation pathways. Such factors are known to those skilled in the art, and usual (statistically common) values, with normal range of values, are to be found in such well-known textbooks as that by C. Kjeldsberg et al., Practical Diagnosis of Hematologic Disorders, Chicago: American Society of Clinical Pathologists Press, 1989. It is also standard practice in the art to combine plasma samples from a large number of normal, healthy volunteer donors to generate a larger "pooled" batch of normal plasma. The advantage of such a pool is that normal plasma samples with identical control values will be available over a long period of time, especially if such standardized control samples are preserved in a frozen or lyophilized (freeze-dried) state.
The term "test sample" or "plasma test sample" as used above and hereinafter simply refers to a plasma sample in which the protein C or protein S content are not yet known, and which is the subject of the coagulometric assay.
It is to be understood that any of the preferred assays of the present invention, when properly set up, can be used to detect the presence or the functional activity of the coagulation-inhibiting proteins, protein C and protein S in a plasma sample; i.e., they can be used to detect or to measure protein C and/or protein S qualitatively or quantitatively, respectively. Thus, the terms "detect" and "detecting", "measure" and "measuring", and "determine" and "determining" as used above and hereinafter are meant to cover both the quantitative and the qualitative aspects of the assays.
In accordance with the present invention, there are also provided several molecular complexes which are novel compositions of matter produced by the process of the assay itself.
FIG. 1 is a line graph which demonstrates that inclusion of soluble thrombomodulin in a coagulometric assay has a significantly different anticoagulant effect on a protein C-deficient plasma sample than it does on a normal control plasma sample. The final amount of soluble thrombomodulin (in micrograms) added to the assay is plotted versus clot time (in seconds) for each sample mixture. The curve with filled circles ( ) represents the normal control plasmas and the open circles (◯) represent the protein C-deficient plasma.
FIG. 2 shows the results of coagulometric assays in which protein S and protein C functional activity are determined using the same methodological embodiment of the present invention. The concentration of normal plasma used to dilute each of the protein C- and protein S-deficient plasma samples is plotted versus clot time (in seconds) for each sample mixture. In this series of studies, an identical amount (about one microgram) of soluble thrombomodulin was added to each assay cup. The filled circles ( ) identify the protein C curve, and the open circles (◯) identify the protein S curve. The point identified by the open triangle (Δ) marks the clot time of a mixture of 50% S-deficient and 50% C-deficient plasma.
FIG. 3 shows the results of a coagulometric assay in which protein S functional activity was determined in a normal plasma sample. The concentration of normal plasma added to a protein S-deficient plasma sample is plotted versus clot time (in seconds) for each sample mixture. In contrast to the data in FIG. 2, the amount of thrombomodulin used in these assays was about 2 micrograms (instead of about 1 microgram) to determine if higher thrombomodulin levels would make the protein S functional assay more sensitive, and therefore more useful, for determining protein S functional activity.
FIG. 4 shows the data from a coagulometric assay in which protein S functional activity was determined in a normal plasma sample to which pure in vitro-activated protein C, but not soluble thrombomodulin, was added at the beginning of the assay. The concentration of normal plasma added to a protein S-deficient plasma sample is plotted versus clot time (in seconds) for each sample mixture.
Novel one-stage coagulometric assay of the present invention.
The following is a detailed description of the novel assay of the present invention useful for directly determining in a plasma sample the functional activities of the vitamin K-dependent coagulation-inhibiting proteins, protein S and protein C. Also provided are descriptions of the necessary reagents and instrumentation used in a preferred embodiment of the present invention. The invention takes advantage of the kinetic environment of an in vitro coagulometric assay to detect the thrombin-mediated coagulation-inhibiting activity of protein C and protein S, and does so, in a preferred embodiment, in an assay in which all of the clotting factors of the intrinsic (in contradistinction to the extrinsic) coagulation pathway are involved, including clotting factors V and Va, and VIII and VIIIa.
Plasma specimen collection and preparation. In preparing plasma specimens for analysis in the coagulometric assay of the present invention, nine parts of freshly-collected patient blood are mixed thoroughly with one part of 3.8% sodium citrate anticoagulant. The anticoagulated blood specimen is then centrifuged for a minimum of 10 minutes at 1000�G to pellet the large cellular components of the blood. The supernatant plasma is removed to a plastic tube, stoppered, and refrigerated at 4�-6� C. until used. Patient plasma is tested within two hours of blood collection to ensure validity and reproducibility of results, and is never held at 37� C. for more than five minutes. Human plasma prepared in a similar manner can also be obtained from American Red Cross blood centers.
Coagulation-activating substance. In performing the coagulometric assay of the present invention, the preferred reagent for use in initiating coagulation in the assay cup is the "DADE�/ACTIN� ACTIVATED CEPHALOPLASTIN REAGENT", commercially supplied by Baxter Healthcare Corporation (Miami, Fla.), and described in the Baxter Healthcare Corporation package insert No. L10293-BH-H (revised 10/88). The active ingredients in this commercial reagent are the negatively-charged organic compound, ellagic acid, and the phospholipid, cephalin, extracted from dehydrated rabbit brain. Use of soybean phospholipid (lecithin) in place of cephalin did not give commercially adaptable results.
Other Reagents used in the one-stage coagulometric assay.
The following reagents are also required for use in the one-stage coagulometric assay of the present invention:
1) saline, prepared as a 0.9% sodium chloride solution in water;
2) soluble thrombomodulin, isolated from rabbit lung tissue and purified according to the procedures of Esmon & Owen (Proc. Natl. Acad. Sci. USA 78: 2249, 1981), and prepared in buffered saline at a final concentration appropriate to the assay; and
3) calcium chloride, prepared as a 25 millimolar solution in water.
The crucial feature of the assay of this invention is the use of soluble thrombomodulin as an additive to the coagulation reaction mixture. The use of soluble thrombomodulin for the activation of protein C in an in vitro coagulometric assay is believed to be unique. When added to the other reagents in the reaction mixture, soluble thrombomodulin provides a highly reactive substrate for the subsequent high-affinity binding of free thrombin. In the presence of calcium, the thrombin in the thrombin-thrombomodulin complex then activates protein C, which, together with protein S, degrades the clot-promoting factors Va and VIIIa in the plasma. Thus, using soluble thrombomodulin anticoagulant as one of the assay reagents, and using protein C-and protein S-deficient plasma samples as deficient plasma controls, the coagulation-inhibiting activities of protein C and protein S can be directly determined in a novel one-stage assay procedure.
As noted above, the coagulometric assay of this invention for protein C and/or protein S is a one-stage assay because. the assay is characterized by a single incubation. In contrast the prior art assays for protein C activate protein C during a prior preactivation step, followed by an incubation to detect the activated protein C formed, and, thus, constitute two-stage assays. Exemplary is the use of copperhead snake venom or "PROTAC-C", discussed above.
In one embodiment of this invention, the active ingredients and other reagents and controls of this invention constitute the components of a commercial assay kit for determining the functional status of vitamin K-dependent coagulation-inhibiting proteins, namely, protein C and protein S. For example, in addition to the soluble thrombomodulin and the coagulation-activating agent of this invention, such a kit optionally contains one or more components selected from (i) a positive control, e.g., a normal plasma sample, (ii) a negative control, e.g., protein C and/or protein S deficient plasma samples, and (iii) a buffered solution containing a calcium salt, e.g., calcium chloride.
Instrumentation used. In a preferred embodiment of the present invention, a Fibrometer™ coagulation timer (Becton, Dickinson and Company Rutherford, N.J.) is used to determine the clot time in the coagulometric assay. When the Fibrometer™ is used, the probes must be thoroughly washed with distilled water and wiped dry with lint-free tissue between tests to prevent carryover of activated plasma proteins. Since there are two different types of probes available for this instrument, a 0.3-ml probe and a 0.4-ml probe, it is necessary to design the assay so that the final volume of reagents added to the reaction cups matches the requirements of the probe which is used.
Coagulation controls. With each series of tests, standard clot time curves are prepared using several control plasma samples in order to ensure the quality, reproducibility and interpretability of the data obtained. The control plasma samples are run in the same manner as the test samples, and establish a range of allowable variation for the control clot time values.
In the present invention, protein C-deficient and protein S-deficient plasma samples, used as factor-deficient plasma controls, were prepared by immunoabsorption of different batches of the same citrated human plasma. Immunoabsorption was carried out using immobilized polyclonal antibodies. For example, to prepare protein C-deficient plasma, one volume of the citrated plasma was mixed in a batch preparation immunoabsorption procedure with nine volumes of anti-protein C coupled to Sepharose�. In contrast, to prepare protein S-deficient plasma, plasma was passed over a polyclonal antibody-Sepharose� affinity column, in a chromatography process well known in the art. Regardless of the immunoabsorption method used, however, it must be noted that the concentration of factors V and VIII may be altered during the processing. Since these factors are crucial in the clotting cascade and are the principal protein targets of activated protein C and protein S as described earlier, alterations in their concentrations will affect the coagulability of and the outcome of assays using the processed plasma samples. Accordingly, the concentrations of factors V and VIII in processed plasma samples must be standardized following immuno-absorption.
Assay Results. The results obtained in the coagulometric assays of the present invention are reported in seconds, and are related to a "normal range" of clot time values obtained in each laboratory. This normal range is established by obtaining plasma samples from a large number of normal healthy donors (for example, from 25 to 40 individuals), and determining the range of statistically common clot time values for these normal donors when tested in the same laboratory. The number of donors needed to establish a statistically acceptable range is determined by each laboratory. Blood is collected by the same method used for collecting test samples from patients, and clot times, using the assay of the present invention, are determined on each normal citrated plasma.
In like manner, a range of values common to populations other than normal, healthy adults is also established as warranted. For example, it may be of interest to establish normal ranges in selected age groups such as pediatric groups or the elderly; or in certain risk groups such as smokers, alcoholics, or individuals on certain medications.
The following examples demonstrate methods and describe protocols useful in practicing the present invention.
In these studies, a Becton-Dickinson Fibrometer� with a 0.4-ml probe was used. The following reagents were placed into a coagulation cup, with care to add them quickly but accurately:
a) 0.1 ml of the "DADE�/ACTIN� ACTIVATED CEPHALOPLASTIN REAGENT" (as described hereinabove);
b) 0.1 ml of normal plasma or protein C-deficient plasma.
As soon as these reagents were added into the cup, a timer was started, the reagents were mixed well, and incubated at 37� C. for exactly 90 seconds. During this brief incubation time, the calcium-independent preactivation of the intrinsic coagulation cascade begins, with the activation of factor XII by kallikrein on negatively charged surfaces, forming factor XIIa, an enzyme which catalyzes the conversion of factor XI, a proenzyme, to its active enzyme form, factor XIa. The rapid preactivation process stops here, in the absence of added calcium ions. At 90 seconds, the following were then added:
c) from 1 to 10 microliters of soluble thrombomodulin (equivalent to 0.1 to 1 micrograms of soluble thrombomodulin, respectively);
d) sufficient volume of prewarmed (37� C.) saline to bring the volume to 0.1 ml;
e) 0.1 ml of prewarmed 25mM CaCl2.
The total volume in the coagulation cup was 400 microliters. Precisely as the calcium chloride was added, the Fibrometer™ timer and probes started automatically. The elapsed time until detection of a fibrin clot was determined automatically by the instrument. The indicated clot time was recorded for each assay cup in a replicate group.
The amount of soluble thrombomodulin added to the coagulometric assay was important. As shown in TABLE 1, the anticoagulant effect of soluble thrombomodulin in the presence of normal control plasma increased in direct proportion to the amount of thrombomodulin added to the assay, The control assay cup which contained only dialyzing buffer, and which lacked thrombomodulin, clotted in 30.4 seconds, whereas the addition of 1 microgram of soluble thrombomodulin increased this clot time more than five fold.
TABLE 1______________________________________Clot time (seconds) in coagulometric assay containingdifferent amounts of soluble thrombomodulin.Thrombomodulin Clot time (seconds)(micrograms) Control Plasma .sup.a Deficient Plasma______________________________________0.1 37.90.2 43.90.6 69.8 64.80.8 112.9 71.81.0 156.9 81.3buffer only 30.4______________________________________ .sup.a Plasma was deficient in protein C
These data also demonstrate that soluble thrombomodulin had a significantly different anticoagulant effect on protein C-deficient plasma than it did on normal control plasma. When these same data were plotted as amount of soluble thrombomodulin added (in micrograms) versus clot time (in seconds), two very different clot time lines were obtained, as shown in FIG. 1.
It is important at this point to realize that the principal difference between the two plasma samples in this test was the presence of functional protein C molecules in the normal plasma sample compared to the absence of these molecules in the protein C-deficient plasma sample. Thus, the difference in slope of the two lines in FIG. 1 reflects primarily the presence or absence of this coagulation-inhibiting protein.
Since soluble thrombomodulin exerts dual anticoagulant functions (one being its heparin-like effect which prevents thrombin from converting fibrinogen into fibrin; and the other being its role in converting protein C into activated protein C, which then degrades factors Va and VIIIa in the coagulation cascade), the clot time curve associated with the protein C-deficient plasma is a reflection solely of the heparin-like activity of thrombomodulin. In contrast, the clot time curve of the normal plasma is a reflection of the combination of the heparin-like activity of thrombomodulin and its protein C activating capacity. Hence, the difference in the clot times between the two curves in FIG. 1 is a novel and simple way to demonstrate and quantify the functional presence of protein C in the normal plasma sample.
A one-stage functional assay of both protein C and protein S was set up essentially the same as in EXAMPLE 1, except that a) a constant amount (1 microgram) of soluble thrombomodulin was added to each assay cup; and b) the normal and deficient plasma samples were mixed together in varying proportions, in a final volume of 100 microliters, to create a series of plasma test samples with different levels of protein C or protein S, from 100% down to 0% of normal. The normal control plasma had full protein C and protein S activity.
The results from these experiments are shown in FIG. 2, in which the filled circles () identify the protein C curve, and the open circles (◯) identify the protein S curve. The point identified by the open triangle (Δ) marks the clot time of a mixture of 50% S-deficient and 50% C-deficient plasma.
The clot time curves for the protein C- and protein S-deficient plasma types were substantially different, as shown. Bearing in mind that the part of an assay dilution curve wherein the assay is most sensitive to a change in the concentration of the measured protein is the region comprising the steepest part of the curve, it is clear that the relative dilutions of protein C- or protein S-deficient plasma at which the coagulometric assays are most sensitive for those factors are substantially different. As shown in FIG. 2, the clot time curve using protein C-deficient plasma is steeper (and therefore more sensitive) in the range of 50% or greater normal plasma than it is in the range of 0 to 50% normal plasma. In contrast, the protein S clotting curve is much steeper (and therefore more sensitive) in the range of from 0 to 25% normal plasma.
These data indicate that:
a) assays to detect protein S functional deficiencies can be satisfactorily performed using dilute preparations of the patient plasma test sample (i.e., small volumes of test plasma relative to the volume of protein-S deficient plasma); and that
b) under the same reaction conditions (i.e., about 1 microgram of thrombomodulin in the assay), assays for the sensitive, detection of protein C functional deficiencies can be performed using small volumes of protein C test plasma mixed into a normal control plasma.
In a more practical application of the present invention, these data also indicate that, under the same react on conditions outlined above (i.e., approximately 1 microgram of thrombomodulin in the assay), assays to detect protein C functional deficiencies permit significantly smaller amounts of protein C-deficient plasma to be mixed into the test plasma sample (i.e. cannot be carried out satisfactorily on dilute preparations of the patient plasma test sample if the diluent is protein C-deficient plasma). Under these conditions, protein C in small amounts of test plasma may be satisfactorily measured by admixture with normal control plasma, thus taking advantage of the sensitive range of protein C detection shown in FIG. 2.
In an effort to improve the sensitivity with which protein S functional deficiencies could be detected, the amount of soluble thrombomodulin in the coagulometric assay of the present invention was raised from 1 microgram (as used above in EXAMPLE 2) to 2 micrograms, as shown in the following example.
Doubling (to 2 micrograms) the amount of soluble thrombomodulin reagent added to the coagulometric assay made the assay more sensitive to determining protein S functional activity. As shown in FIG. 3, a substantial range of clot times (from approximately 90 seconds to 200 seconds; a range of about 110 seconds) was observed when protein S was assayed at concentrations varying from 0% (equivalent to 100% protein S-deficient plasma) to 25% of normal (equivalent to 3 parts protein S-deficient plasma to 1 part normal plasma). Thus, by adding about 2 micrograms of soluble thrombomodulin to the assay system, the assay became substantially more sensitive to protein S in the test samples than was detected using only 1 microgram of soluble thrombomodulin. Therefore, the sensitivity with which protein S functional deficiencies in plasma are detected is dependent on the concentration of thrombomodulin used in the assay.
It was important to compare these results with those of an assay for protein S based on adding exogenous, preactivated protein C into a coagulometric assay. The following example demonstrates that, in the absence of soluble thrombomodulin, the results were substantially different and were not as useful as the results obtained from assays which incorporated soluble thrombomodulin.
The data in FIG. 4 were obtained in an assay which utilized activated protein C, but not soluble thrombomodulin, as a coagulation-inhibiting protein factor. In a Fibrometer™ coagulation cup, the following were added:
a) 0.1 ml of "DADE�/ACTIN� ACTIVATED CEPHALOPLASTIN REAGENT" described in detail above;
b) 0.1 ml of plasma; and
c) 0.4 micrograms of activated protein C.
These reagents were incubated at 37� C. for exactly 90 seconds, at which time 0.1 ml of saline was added, followed immediately by 0.1 ml of calcium chloride, activating the clotting reaction and the instrument timer.
The data show that the range in clot time (from approximately 54 seconds with undiluted protein S-deficient plasma, to about 89 seconds with 75% protein S-deficient plasma; a range of only about 35 seconds) was approximately one-third of the range in clot time shown in FIG. 3 in the previous example. Because the change in reagents reduced the range of assay responses by nearly 67%, it is clear that adding activated protein C instead of soluble thrombomodulin into the coagulometric assay did not provide as useful a measure of protein S function as was found in the coagulometric assay of the previous examples.
It will be appreciated that changes may be made in the nature, composition, operation and arrangement of the various elements, steps and procedures described herein without departing from the spirit and scope of the invention as defined in the following claims.
|Cited Patent
|Filing date
|Publication date
|Sep 23, 1985
|Apr 5, 1988
|Boehringer Ingelheim International Gmbh
|Blood coagulation inhibiting proteins, processes for preparing them and their uses
|Jan 15, 1987
|May 31, 1988
|Kowa Co., Ltd.
|Thrombin-binding substance and process for preparing the same
|May 9, 1986
|Jul 18, 1989
|Pentapharm Ag
|Protein C activator, methods of preparation and use thereof
|Oct 4, 1988
|Mar 19, 1991
|Boehringer Mannheim Gmbh
|Process for photometric determination of protein C and/or protein S
|Aug 21, 1989
|Sep 24, 1991
|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
|Jun 14, 1989
|Jun 9, 1992
|Oklahoma Medical Research Foundation
|Factor xa based anticoagulant compositions
|Aug 4, 1989
|Sep 15, 1992
|Oklahoma Medical Research Foundation
|Inhibition of tumor growth by blockade of the protein C system
|Jun 24, 1991
|Jan 18, 1994
|The Scripps Research Institute
|Activated protein C polypeptides and anti-peptide antibodies, diagnostic methods and systems for inhibiting activated protein C
|Jun 17, 1992
|Jul 19, 1994
|Immuno Aktiengesellschaft
|Method of preparing activated protein C
|Apr 6, 1993
|Jun 20, 1995
|The Trustees Of Columbia University In The City Of New York
|Calreticulin: a novel antithrombotic agent
|Jul 4, 1990
|Aug 8, 1995
|Chromogenix Ab
|Method for determining the functional activity of free protein S or protein C in a plasma sample
|May 13, 1992
|Aug 22, 1995
|Dahlbaeck; Bjoern
|Method for the diagnosis of blood coagulation disorders
|Jul 9, 1993
|Sep 26, 1995
|Eli Lilly And Company
|Protein C derivatives
|Faioni et al, "Blood"; vol. 71(4), pp. 940-946, (Apr. 1988).
|Faioni et al, Blood ; vol. 71(4), pp. 940 946, (Apr. 1988).
|Citing Patent
|Filing date
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|Nov 27, 1996
|Jun 16, 1998
|Ahs Hospital Corp.
|Factor V ratio blood test for susceptibility to thromboembolism
|Apr 30, 1997
|Sep 14, 1999
|Medtronic, Inc.
|Platelet function evaluation technique for citrated whole blood
|Apr 30, 1997
|Jan 4, 2000
|Medtronic, Inc.
|Apparatus for performing a heparin-independent high sensitivity platelet function evaluation technique
|Oct 28, 1999
|Mar 20, 2001
|Actv, Inc.
|Compressed digital-data interactive program system
|Oct 28, 1999
|Apr 10, 2001
|Actv, Inc.
|Compressed digital-data interactive program system
|Jan 4, 1999
|Apr 24, 2001
|Medtronic, Inc.
|Method for determining platelet inhibitor response
|Jun 11, 1999
|May 15, 2001
|Medtronic, Inc.
|Platelet function evaluation technique for citrated whole blood
|Feb 12, 1998
|Jun 12, 2001
|University Of Medicine And Dentistry Of New Jersey
|Rapid assessment of the coagulant activity of blood
|Oct 28, 1999
|Jun 26, 2001
|Actv, Inc.
|Compressed digital-data interactive program system
|Sep 13, 2000
|Aug 13, 2002
|Affinity Biologicals, Inc.
|Method for measuring antithrombin activity
|Mar 16, 2001
|Apr 29, 2003
|Medtronic, Inc.
|Method for determining platelet inhibitor response
|Mar 16, 2001
|Apr 29, 2003
|Medtronic, Inc.
|Apparatus for performing a platelet inhibition test
|Mar 16, 2001
|Apr 29, 2003
|Medtronic, Inc.
|Method for determining a contact activator for platelet activation
|Dec 19, 2001
|Feb 15, 2005
|Instrumentation Laboratory Company
|Protein S functional assay and kit therefor
|Dec 21, 2001
|May 9, 2006
|T.A.C. Thrombosis And Coagulation Aktiebolag
|Methods and reagents for determining protein S
|Jan 28, 1994
|Jan 30, 2007
|T.A.C. Thrombosis And Coagulation Ab
|Assays for determining anticoagulant cofactor activity
|Dec 15, 2004
|Dec 23, 2008
|Instrumentation Laboratory Corporation
|Protein S functional assay
|May 11, 2005
|Apr 20, 2010
|Medtronic, Inc.
|Point of care heparin determination system
|Sep 21, 2004
|Jun 1, 2010
|The University Of North Carolina At Chapel Hill
|Soluble phospholipids for use in clotting factor assays
|Nov 19, 2008
|Dec 18, 2012
|Instrumentation Laboratory Company
|Kit for protein S functional assay
|Mar 29, 2010
|Aug 12, 2014
|Medtronic, Inc.
|Point of care heparin determination system
|Apr 21, 2003
|Nov 6, 2003
|Dade Behring Marburg Gmbh
|Method for determining the anticoagulatory potential of a sample
|Dec 15, 2004
|May 5, 2005
|Instrumentation Laboratory Company
|Protein S functional assay
|May 11, 2005
|Jan 26, 2006
|Wei Qin
|Point of care heparin determination system
|Sep 21, 2004
|Feb 15, 2007
|University Of North Carolina At Chapel Hill
|Soluble phospholipids for use in clotting factor assays
|Nov 16, 2006
|Sep 13, 2007
|Dahlbaeck Bjoern
|Assays for Determining Anticoagulant Cofactor Activity
|Nov 19, 2008
|Mar 26, 2009
|Yong Dai
|Protein S Functional Assay
|Jul 21, 2008
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|Viridis Biopharma Pvt. Ltd.
|Treatment of human disease conditions and disorders using vitamin k analogues and derivatives
|Oct 7, 1998
|May 12, 1999
|Dade Behring Marburg GmbH
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|Sep 21, 2004
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|Univ North Carolina
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|U.S. Classification
|435/13, 436/63, 435/4, 435/69.6, 436/69, 514/833
|International Classification
|Cooperative Classification
|European Classification
|Jan 2, 1998
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Mouna Raagam Trailer (1986)
15 August 1986 Drama, Romance 160 mins
Divya, a woman grieving over the death of her lover, is convinced into an arranged marriage with Chandra Kumar. Over a year the couple attempt to adjust to each other's presence and live with each other. Revathi is forced to marry Mohan in an arranged marriage. She lives in the memory of her lost love Karthik and asks for a divorce. The law requires them to stay together for a year before granting the divorce. That gives time for a lot of incidents in their lives and things change.
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text: Agent Based Software Testing Framework (ABSTF) for Application MaintenanceK. Karnavel, V. Divya, Gnanakeerthika, P. KarthikaArXiv2013Software testing framework can be stated as the process of verifying and validating that a computer program/application works as expected and meets the requirements of the user. Usually testing can be done manually or using tools. Manual testing can be time consuming and tedious, also it requires heavy investment of human resources. Testing tools in fact… (More)


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The global burden of disease and challenges of safe and accessible health services means that more than ever practitioners and researchers need skills for developing, testing, evaluating and implementing evidence-based health care in highly complex situations. This programme provides practitioners with the skills and knowledge to tackle these challenges.
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The University of Exeter invests significantly in a range of funding opportunities for talented prospective postgraduates. Please visit: Funding for further details. Here you can search our database for funding opportunities suitable for your area of interest.
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Hi There! Is this Your First Time?
Did you know if you Register you have access to free search tools including the ability to save listings and property searches? Did you know that you can bypass the search altogether and have listings sent directly to your email address? Check out our how-to page for more info.
- Price$1,200,000
- Beds4
- Baths4
- Sq. Ft.6000-6199
- Acres0.36
- Built2006
1125 Dickson Road, Campobello SC, 29322 MLS® #1336201
Home in None Campobello SC
Water front home on Lake Bowen with amazing mountain views! One of a kind design with attention to detail throughout. Stunning contemporary home has over 6,000 square feet within its two stories and basement areas. A dramatic entry foyer creates an edgy and inviting space for welcoming family and friends. The is naturally lit by its 114 panels of glass and offers views of the water and mountains from every level. All levels of the home can be accessed by the custom design glass elevator or the metal staircase design by renowned Heirloom Stairs. A great setting for entertaining can be found in the open kitchen and greatroom areas. Custom, German designed kitchen cabinetry with granite counter-tops offer a modern design that's second to none. The family or friends will gather around the over-sized kitchen island while they watch the chef of the house prepare meals on Wolfe gas range with pot filler, steamer, and sub zero refrigerator. Art gallery on the main level offers a great conversation area for guests and family as well. Main level master suite has amazing views of the water and mountains. Master bath offers an over-sized tile shower with block glass walls, farm sink, heated floors, and a custom designed walk-in closet. The second story of the home is specifically designed for relaxation. Working in the main level home office will feel like vacation with these views. Whether you choose the inside venue of the sitting area or the covered rear deck, you won't be disappointed in the breathtaking views. The lower level offers space for guests to relax and unwind. The rec. room on the lower level has a more relaxed atmosphere as it opens to the rear patio that stretches the length of the house. You won't believe the custom cabinets and built-ins that the lower level office/craft room has to offer. The three guest rooms all have walk-in closets and great access to two full baths. 2 story ceilings, all fixtures, and finishes are unique and perfectly in tune with the contemporary design. Entertain outside on the upper patio,k lower patio with fire pit, or dock areas. Hard coat stucco exterior and block and iron interior structure form a sturdy and low maintenance home. Two HVAC units and water heaters. For a private showing, contact the listing agent.
Essential Information
- Price$1,200,000
- Bedrooms4
- Bathrooms4.00
- Full Baths3
- Half Baths2
- Square Footage6000-6199
- Acres0.36
- Year Built2006
- TypeResidential
- Sub-TypeSingle Family-Detached
- StyleContemporary
- StatusActive
- AmenitiesNone
- ParkingNone
- Garage CapacityNone
- GaragesNone
- Is WaterfrontYes
- Green CertificationOther - Docs Req
- ExteriorMasonry Stucco
- Exterior FeaturesDeck, Patio, Porch-Front, Porch-Other, Tilt Out Windows, Windows-Thermal, Sprklr In Grnd-Full Yard
- Lot DescriptionLevel, Mountain View, Water Access, Water Front, Water View, Dock
- RoofMetal
- FoundationBasement
Listing Details
- Listing Agent:Mitzi Kirsch
- Listing Office:Century 21 Blackwell
Community Information
- Address1125 Dickson Road
- SubdivisionNone
- CityCampobello
- CountySpartanburg
- StateSC
- Zip Code29322
- InteriorCeramic Tile
- Interior Features2 Story Foyer, Cable Available, Ceiling 9ft+, Ceiling Fan, Countertops-Solid Surface, Open Floor Plan, Security System Owned, Smoke Detector, Some Window Trtmnts
- AppliancesCook Top-Gas, Dishwasher, Disposal, Refrigerator, Ice Machine
- HeatingElectric, Forced Air, Multi-Units
- CoolingCentral Forced, Electric, Multi-Units
- FireplaceYes
- FireplacesNone
- Stories2+Basement
- FloorsCeramic Tile
School Information
- ElementaryCampobello-Gramling
- MiddleCampobello-Gramling
- HighChapman
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Friday 22 December 1989
It was Friday, under the sign of Capricorn. The US president was George H. W. Bush (Republican).
Famous people born on this day include Logan Huffman and Jharal Yow Yeh.
In that special week of December people in US were listening to Another Day In Paradise by Phil Collins.
In UK You Got It (The Right Stuff) by New Kids On The Block was in the top 5 hits.
Always, directed by Steven Spielberg, was one of the most viewed movies released in 1989
while Roseanne by Roseanne Barr was one of the best selling books.
On TV people were watching The Simpsons.
If you liked videogames you were probably playing Break In or Bad News Baseball.
But much more happened that day: find out below..
You can also have a look at the whole 1989 or at December 22 across the years.
Old Newspapers
Have a look at the old newspapers from 22 December 1989 and get them!
TV Series
Which were the most popular TV series released in the last months ?
Which were the most popular movies released this week ?
Find out your future
Get a FREE Numerology report based on the digits of 22 December 1989!
Wish him/her..
Historical Events
Which were the important events of 22 December 1989 ?
- R.C. Saints - commemoration of Frances Xavier Cabrini
- Spain: the main draw of Spanish National Lottery, where it is a Christmas tradition.
- Astrology: First day of sun sign Capricorn
- In the Northern Hemisphere, the winter solstice occurs on or very close to this date. In the Southern Hemisphere, the summer solstice occurs around this time.
- Also see December 22 (Eastern Orthodox liturgics)
- Japan - T?ji (winter solstice)
Famous Birthdays:
- Logan Huffman: American actor.
- Jharal Yow Yeh: professional rugby league player.
- Jordin Sparks: American singer and American Idol winner.
- Beautyklove : YouTube Star.
- Jharal Yeh: Australian rugby player.
Famous Deaths:
- Samuel Beckett: Irish author of prose, fiction and avant-garde plays, who established the contemporary theater of the absurd with his surrealistic "Waiting for Godot.
- After 23 years of dictatorial rule, Romania ousts Nicolea Ceausescu
- Chad adopts its constitution.
- After a week of bloody demonstrations, Ion Iliescu takes over as president of Romania, ending Nicolae Ceausescu's Communist dictatorship.
- Berlin's Brandenburg Gate re-opens after nearly 30 years, effectively ending the division of East and West Germany.
- US cold wave: -42 degrees F in Scottsbluff, Nebraska, -47 degrees F in Hardin, Montana and -60 degrees F in Black Hills, South Dakota.
Featured Contents
Would you like to write an original contribution on a topic related to this specific date and get featured on Takemeback.to ? Send us your proposal!
Music Charts
Which were the top hits in that special week of December 1989 ?
Top #10 songs in the USA
- Phil Collins - Another Day In Paradise
- Linda Ronstadt - Don't Know Much
- Billy Joel - We Didn't Start The Fire
- Janet Jackson - Rhythm Nation
- Taylor Dayne - With Every Beat Of My Heart
- Soul II Soul - Back To Life
- Technotronic - Pump Up The Jam
- Cher - Just Like Jesse James
- New Kids On The Block - This One's For The Children
- Bon Jovi - Living In Sin
Search For Your Ancestors
Subscribe to our Newsletter
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...and if 22 December 1989 was your Birth Date then Join our Birthday Club!
Make this date unforgettable
Genealogy: find out your family heritage
Magazine Covers
What news were making the headlines those days in December 1989?
- Time: TIME Magazine Cover: Money Laundering -- Dec. 18, 1989
- SportsIllustrated: Sports Illustrated: no.9204
- Time: Building a New World'
Which were the most popular books released in the last weeks ?
Remember when
What videogames would you be playing back in the days ?


text:
A beautiful design from Marc by Marc Jacobs. This stainless steel time piece features a round case with a white dial. The attractive dial shows off elegant silver hour markers with four markers in letter form branding MARC from 12 to 3 o'clock. It has silver hands and a small dial with the second hand. This watch fastens with a silver metal bracelet and is powered by a quality quartz movement.
JB Watches accept all Credit cards during checkout and guarantee safety to all of their customers. In case of an issue, please contact the customer services team.
How long will it take to receive my order?
We always aim to process every order within 24 hours of it being placed
UK delivery – From next business day up to one week.
International – Varies depending on destination.
Click here for more information
Who will deliver my order?
To allow us to offer you the most competitive prices, we use a range of established couriers including Royal Mail, Interlink/DPD, MyHermes, and Parcel Force.
If you are not happy with your item you can always return it to us within 30 days of purchase


text: The PNEUMATIK BERLIN GmbH is an established technology company and a valued partner in medical technology. Offers complete solutions, from consulting, planning and development to delivery, installation and commissioning. The continuous support of customers via the company's own or sub-contracted service and a wide network of dealers. In addition to the aspects of functionality, compatibility, ergonomics and design have the highest quality and safety a top priority.


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Couldn't help but notice that the Qur'an claims Jews worship Ezra
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McDonald's? Really? Nothing against McDonanld's, but that buliding is a historical landmark. This is coming from a Coventry area, Cleveland Hts resident. One of the things that makes Cleve Hts so unique are the older houses and buildings that are well kept and preserved, giving our suburb and very unique character. Surely, you can renovate the theatre or think of something else unique to put there, that would be successful there, besides a McDonalds. When this recession is over, you'll be looking at that McDonald's kicking yourself, b/c of your knee jerk reaction.
I hope it doesn't have any high fructose corn syrup or modified food starch in it. If not, I'm game!


text: In this blog post, we'll look at some tips on how to configure Aurora RDS parameters. I was recently deploying a few Aurora RDS instances, a process very similar to configuring a regular RDS instance. I noticed a few minor differences in the way you configure Aurora RDS parameters, and very few articles on how […]Read More


text:
The Albion striker is a target for Queens Park Rangers who have already had one offer rejected - believed to be £2m.
But Garlick insists West Brom have no need to sell any of their squad, and it will require substantially high offers for the club to change their stance.
"Last summer there were supposedly players leaving but we kept hold of most of them," Garlick said. "We don't have to sell any of our players. It's only on our terms.
"It's well documented about Peter. We had an offer and we don't intend to sell any of our players.
"When an offer comes in, we speak to the player out of courtesy. We've had that chat with Peter and he knows the situation."
Albion rejected a £5m bid, also from Rangers, for defender Jonas Olsson who only signed a new four year contract in October.
And Garlick admits does not expect any new players to arrive at the Hawthorns before the transfer window closes a week today.
"We're happy with the squad we've got and there are no immediate plans to bring anyone in," Garlick added.
"If an opportunity presents itself, then we will look at that, but we're looking more towards the summer."
Source: Sports.naija.ng


text:
A dedicated Colne manageress will be paying a touching tribute to her grandma and late father-in-law when she runs the Blackpool Autumn Breaker 10K in October.
By taking on the challenge, Sharon Jackson (33) aims to raise awareness and money for the Alzheimer's Society.
The mum-of-two, who runs Colne business Harp Ceramics with her husband Paul, has personally experienced the tireless efforts of the charity – as her grandma Kathleen is currently suffering with dementia, while her father-in-law Colin Jackson died from Alzheimer's at just 57.
She will be completing the challenge with her brother Neil.
Sharon,of North Valley Road, said: "We are trying to raise as much awareness as possible.
"People think that Alzheimer's is just linked to older people and losing your memory.
"Paul's dad was still fully aware of who he was, but he started losing his speech and sight, and he was bed-ridden.
"It is a brain condition, and Colin's brain started dying before his memory did.
"The Alzheimer's Society have been superb from start to finish."
Although Sharon only started running in April, she is now hopeful to complete more 10K treks and half marathons for the Alzheimer's Society and Pendleside Hospice.
Anybody wanting to make a donation can go to her JustGiving page or visit her at Harp Ceramics, in North Valley Road.
Sharon, a former manageress of Debenhams, and Boundary Mills, added: "I would be so grateful if people could spare a couple of pence.
"It doesn't matter if it is 2p or 10p – anything is better than nothing.
"I'm really excited for the 10K, I can't wait.
"I didn't do any running up until April, and it's really addictive and you get fit really quick."
To make a donation visit www.justgiving.com/Sharon-Jackson5


text:
|13 Aug 2012
BSOD while doing anything, AMD 955 x4 machine, windows 7 ultimate 64bi
I have a AMD 955 x4 machine here with 4GB. I don't know if it's a memory or driver problem. It could very well be a driver error but I don't know how to find out which.
I ran the program and here is my attached zip file. If someone could please show me what's wrong I would be grateful.
|My System Specs
|13 Aug 2012
Windows 7 Enterprise x64, Windows 8 Enterprise x64
Update your driver for "NETGEAR WPN311 RangeMax(TM) Wireless PCI Adapter".
fffff880`05646000 fffff880`0579e000 athrx Tue Jun 09 20:06:49 2009 (4A2EA4B9)
|My System Specs
|13 Aug 2012
Thanks Tobias. For now I just diabled it. Crossing my fingers. Thanks again.
|My System Specs
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Code enforcement keeping an eye on SXSW events
Austin Police say the number of South by Southwest event applications has increased by 50 percent during the last two years. City officials are working to make sure everyone is playing by the rules.
"A huge light show, disco ball, smoke, all that good stuff," says Eddie Dunn, general manager of Ten Oak.
It takes a big production to throw a SXSW event. Dunn is working around the clock to make it happen.
"This room will look completely different tonight. So about 6:00 p.m. we'll start converting it. For every band, they have different needs, so that gives us time to prepare for that," says Dunn.
They've already had two days of private events. So far, Dunn says he's seen more enforcement.
"We kind of let too many people in, so we kind of had to stop and move some people out. It gets crowded," says Dunn.
This year 205 event applications were processed for SXSW. That is 50 percent more than two years ago.
"For a club, empty floor space and a stage is where the people can be. Based on that square footage, we can figure out a certain number of people they could have for their capacity. Now if you take that same space and put a bunch of tables and chairs in there, the same number of people wouldn't fit," says Division Chief Rob Vires, Fire Marshal, Austin Fire Department.
If a venue looks like it's reaching capacity, or is slightly over, code enforcement will recommend options such as one in, one out. But, different measures are taken if a venue is way over the limit.
"We'll write a citation. We've also closed clubs. We closed one down so far. They had an occupant-load card missing. Their occupancy should have been 279 and they had over 400 people inside the club. That wasn't safe," says Vires.
They've had to manage about a dozen overcrowding situations. Division Chief Rob Vires pointed out the Ghost Ship Fire last year in Oakland California. The warehouse was hosting a concert when all of a sudden a fire broke out, killing 36.
"We don't want that sort of situation here. So the idea is to try to maintain crowds and make sure that proper permitting and exiting is available for the number of folks inside. So that if God forbid there were an incident, that people would have the ability to get out safely and not be trapped inside," says Vires.
Ten Oak will have musical acts performing Wednesday through Sunday. They have hired more staff to handle the large crowds.
"First 350 in, come on in. After that, it's going to be one in, one out. So we'll reach that capacity and that's it," says Dunn.
Code enforcement says they want to work with the organizers, the last thing they want to do is shut down an event.


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Pre-Labor Contractions
(Braxton Hicks)
Also try a bath as soaking in a tub of water helps to push fluids into the blood stream. And rest if you had an active day. Your body is already doing so very much so please take care when your active and pregnant.
Read about nutrition and it's role in pregnancy and preterm labor at (www.blueribbonbaby.org)
Labor Contractions
(Let's have a baby!)
Blimey, I could have done with this list when I had my second!
when i walk the "brackston hicks" get stronger an hurt sometimes an i get them sometimes even when i move posistions but they are ireg an dont amke me stop what im doing or anything my whole stomach will tighten an sometimes last for aminute or so but they dont hurt are thesee early labor contractions ( like are thes the ones that dialte my cervix? or are they jsut brackston hicks.. ? im so confused
im 2cm an 90% effaced an lost my plug
Re: Toning Pre-Contractions vs Labor Contractions
Thanks for the list! I am also thinking -- hopefully I will just know b/c it will feel different.
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Heretic Trailer (2012)
"Some Sins can never be Forgiven" 05 November 2012 Horror 94 mins
A troubled Catholic priest, Father James, finds his faith crushed when a young girl he promises to protect commits suicide. Months after her death, he is forced to return to his old parish and to the scene of her suicide, a derelict mansion house. Trapped in the house overnight James becomes convinced that he is being haunted by the ghosts of the girl and her dead Stepfather, risen from the grave to seek a blood retribution for the awful tragedy that he allowed to take place
Watch the official Heretic 2012 trailer in HD below or find more Heretic videos on Vidimovie.


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Saturday, July 2, 2011
So fresh & so clean
The most beautiful crisp winter saturday morning for a soy latte & sanoma toast... then onto cruise the bondi farmers markets.. Filled with fresh clean goodies, organic veges and cute little families.. Oh, not to forget about the vegan treats and the fresh flowers!
I highly advise you pay a visit! Saturday mornings 10am-1pm.
On another note, I spent last night wining & dining at Billy Kwong with a special friend of mine, Kinae... It was so so amazing. The food was to-die-for and the service was beyond memorable! 5 stars for Billy K!
Posted by Karla at 3:01 PM
2 comments:
- Jazzy E (hivenn) said...
love these! x hivenn
- July 6, 2011 at 9:26 PM
- Candice Rose said...
Just around the corner!!!!
- July 27, 2011 at 1:31 PM
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