Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-08-12
2002-07-09
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007920, C435S007100, C435S006120, C530S350000, C530S300000, C514S002600
Reexamination Certificate
active
06416963
ABSTRACT:
BACKGROUND OF THE INVENTION
The clotting of blood is part of the body's natural response to injury or trauma. Blood clot formation derives from a series of events called the coagulation cascade, in which the final steps involve the formation of the enzyme thrombin. Thrombin converts circulating fibrinogen into fibrin, a mesh-like structure which forms the insoluble framework of the blood clot. As a part of hemostasis, clot formation is often a life-saving process in response to trauma and serves to arrest the flow of blood from severed vasculature.
The life-saving process of clot production in response to an injury can become life-threatening when it occurs at inappropriate places in the body. For example, a clot can obstruct a blood vessel and stop the supply of blood to an organ or other body part. In addition, the deposition of fibrinogen contributes to partial or complete stenosis of blood vessels, resulting in chronic diminution of blood flow. Equally life-threatening are clots that become detached from their original sites and flow through the circulatory system causing blockages at remote sites. Such clots are known as embolisms. Indeed, pathologies of blood coagulation, such as heart attacks, myocardial infarctions, strokes, and the like, have been estimated to account for approximately fifty percent of all hospital deaths.
Fibrinogen is synthesized and secreted into the circulation by the liver. Circulating fibrinogen is polymerized under attack by thrombin to form fibrin, which is the major component of blood clots or thrombi. Subsequently, fibrin is depolymerized under attack by plasmin to restore the fluidity of the plasma. Many of the steps in the polymerization and depolymerization processes have been well established, Doolittle et al.,
Annu. Rev. Biochem
., 53:195-229 (1984). The elevated levels of fibrinogen which are part of the acute phase response occurring in the wake of infections and trauma are now known to come from increased hepatic production, primarily in response to interleukin-6 (IL-6). Seghal et al.,
Ann N.Y. Acad. Sci
. 557:1-583.
Fibrinogen, one of the more well-studied proteins, plays a central role in clot formation and wound healing. It has a complex structure which includes a heavily disulfide-bonded hexamer composed of two copies each of the &agr;, &bgr; and &ggr; subunits. Recently, new attention has been given to structure/function relationships in the fibrinogen molecule. This new interest has in part been prompted by growth in the understanding of this protein's range of activity in normal and pathological states, see for example, Blomback et al., Biotechnology of Blood, 225-279 (1991), Bini et al.,
Ann N.Y Acad. Sci
., 667:112-126 (1992) and Dvorak et al.,
Ann N.Y. Acad. Sci
., 667:101-111 (1992).
By the late 1960's, the general subunit structure of fibrinogen was firmly established. Blomback et al.,
Nature
218:130-134. A decade later, the complete amino acid sequence was reported. Lottspeich et al.,
Hoppe-Seyler's, Physiol. Chem
. 358:935-938 (1997), Henschen et al.,
Hoppe-Seyler's, Physiol Chem
., 358:1643-1646, Henschen et al.,
Hoppe-Seyler's, Physiol Chem
., 360:1951-1956, Doolittle et al.,
Nature
, 280:464-468 (1979). Over the next 10 years, the cluster of three separate genes encoding the &agr; (alpha), &bgr; (beta) and &ggr; (gamma) subunits was identified on chromosome 4q23-q32, Kant et al.,
Proc. Natl. Acad. Sci. USA
, 82:2344-2348 (1985), and the apparently complete genetic sequences of all three fibrinogen subunits were published. Chung et al.,
Adv. Exp. Med. Biol
., 281:39-48 (1991). These studies indicated that the a subunit lacked a globular C-terminal domain comparable to those present in the &bgr; and &ggr; subunits.
The subsequent discovery of an additional exon (i.e., exon VI) downstream from the established a subunit gene has resolved the evolutionary mystery posed by the imperfectly parallel structure of the three major subunits. Fu et al.,
Biochemistry
, 31:11968-11972 (1992), Weissbach et al.,
Proc. Natl. Acad. Sci. USA
, 87:5198-5202 (1990). A novel fibrinogen &agr; chain transcript has been identified at low frequency bearing the exon VI-derived sequences as a separate open reading frame. Additional splicing leads to the use of this extra sequence to elongate the &agr; chain by 35% (236 similar to those of the &bgr; and &ggr; chains.
A major impetus to fibrinogen research has been provided by the recent identification of this long overlooked, naturally occurring elongated version of the &agr; subunit, designated “&agr;
E
”. See Fu et al.,
Biochemistry
, 31:11968-11972 (1992). Evidence shows that the &agr;
E
chain is assembled into fibrinogen molecules and that its synthesis is enhanced by interleukin-6 (IL-6). These facts suggest that the &agr;
E
subunit participates in both the acute phase response and in normal physiology.
Using a polyclonal rabbit antibody preparation specific to the VI-domain or &agr;
E
C domain, &agr;
E
was demonstrated to occur in plasma fibrinogen as part of (&agr;E&bgr;&ggr;)
2
, a homodimeric (i.e., symmetrical) molecule of 420 kilodaltons (kDa). Fu et al.,
Proc. Natl. Acad. Sci. USA
, 91:2625-2628 (1994). This species has been designated “fibrinogen-420” (&agr;
E
&bgr;&ggr;)
2
to distinguish it from the abundant 340 kDa form of fibrinogen, denoted “fibrinogen-340”(&agr;&bgr;&ggr;)
2
). Fibrinogen-420 accounts for approximately 1% of the total fibrinogen in normal adult plasma and 3% of the total in umbilical cord plasma. Grieninger et al.,
Blood
, 90:2609 (1997). The relatively low circulating level of fibrinogen-420 is undoubtedly responsible for its having escaped detection. These two &agr;
E
C domains that distinguish Fibrinogen-420 from Fibrinogen-340 are likely to significantly influence the fibrinogen molecule's multiple binding capacities and functions.
Transcripts encoding fibrinogen subunit counterparts having exceptionally high C-terminal homology to human &agr;
E
have been detected thus far in lamprey, where it arises from a second &agr; gene, as well as in chicken, rabbit, rat, and baboon. See Pan et al.,
Proc. Natl. Acad. Sci. USA
, 89:2066-2070 (1992), Doolittle et al.,
Thromb. Res
., 68:489-493 (1992) and Fu et al.,
Genomics
30:71-76 (1995). This degree of &agr; subunit-associated globular domain preservation in the vertebrate genome signals an important, if as yet unknown, role for &agr;
E
. Clues to its potential significance may lie in the similarity of the extension in &agr;
E
, not only to the corresponding regions of the fibrinogen and chains, but also to carboxy domains of a number of non-fibrinogen proteins from fruit fly to man. Chung et al.,
Biochemistry
, 22:3244-3250 (1983), Chung et al.,
Biochemistry
22:3250-3256 (1983), Baker et al.,
Science
250:1370-1377 (1990), Koyama et al.,
Proc. Natl. Acad. Sci. USA
, 84:1609-1613 (1987), Morel et al.,
Proc. Natl. Acad. Sci. USA
, 86:6582-6586 (1989), Nies et al.,
J. Biol. Chem
., 266:2818-2823 (1991), Norenberg et al.,
Neuron
, 8:849-863 (1992), Xu et al.,
Proc. Natl. Acad. Sci. USA
, 87:2097-2101. Where functions are known, these non-fibrinogen proteins are constituents of the extracellular matrix and have adhesive properties. It is expected that continued research will permit the determination of whether the &agr;
E
globular domain contributes in a subtle way to the primary function of fibrinogen (clot formation and wound healing) or, following the example of other differentially used exons, promotes an alternative function. Chan et al.,
Science
, 254:1382-1385 (1991), Descombes et al.,
Cell
, 67:569-579 (1991), Early et al.,
Cell
, 20:313-319 (1980). Thus there is a need to isolate fragments of the Fibrinogen-420 molecule.
In clinical settings it is commonly desirable to activate or potentiate the fibrinolytic system. This is particularly necessary in cases of myocardial infarction in which coronary arteries become occluded and require recanalization. Catheterization has proven somewhat effective in such recanalization, but pharmacologic age
Applegate Dianne
Grieninger Gerd
Stoike-Steben Lara
Carlson Karen Cochrane
Hoffmann & Baron , LLP
New York Blood Center
Robinson Hope A.
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