In vitro methods for determining in vivo thrombotic events

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...

Reexamination Certificate

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C435S007210, C435S007240, C435S242000, C435S007920, C435S007930, C424S811000, C424S185100, C530S387900, C530S350000, C530S300000, C436S518000, C436S007000, C436S548000

Reexamination Certificate

active

06787320

ABSTRACT:

TECHNICAL FIELD
The present invention relates to a fragment of the C-terminal region of GPIIb heavy chain (hGPIIb) that is produced in vivo by indigenous protease cleavage of the GPIIb-IIIa platelet glycoprotein receptor, and to in vitro detection of the fragment in body fluids of individuals undergoing thrombotic events. The present invention also relates to polypeptide analogs of the hGPIIb C-terminal fragment, and antibodies that immunoreact with the C-terminal fragment and to diagnostic methods for detecting thrombi and clotting disorders.
BACKGROUND
Thrombosis involves cell adhesion of platelets. Platelet cell adhesion generally involves recognition of specific adhesive proteins by the platelet cell surface receptor GPIIb-IIIa.
GPIIb-IIIa is a noncovalent, Ca
++
dependent, heterodimer complex comprised of noncovalently associated alpha and beta subunits. Jennings et al.,
J. Biol. Chem.,
257:10458-10466 (1982). The alpha subunit, GPIIb consists of a heavy chain (hGPIIb) having a relative molecular weight of about 120 kilodaltons (KDa), and a light chain (lGPIIb) of about 20 KDa that are linked together by disulfide bonds. The beta subunit, GPIIIa is a single chain polypeptide of about 100 KDa. Phillips et al.,
J. Biol. Chem.,
252:2121-2126 (1977).
GPIIb-IIIa contributes to platelet function through interactions with RGD-containing proteins such as fibrinogen [Bennett et al.,
Proc. Natl. Acad. Sci. USA,
80:2417-2421 (1983)], fibronectin [Ginsberg et al.,
J. Clin. Invest.,
71:619-624 (1983)], and von Willebrand factor [Ruggeri et al.,
Proc. Natl. Acad. Sci. USA,
79:6038-6041 (1982)], and therefore is a component of the common platelet adhesive protein receptor [Pytela et al.,
Science,
231:1559-1562 (1986) and Plow et al.,
J. Biol. Chem.,
259:5388-5391 (1984)].
The sites on GPIIb-IIIa that are required to function as an adhesion receptor are not well characterized. Several observations suggest that a functionally significant site on GPIIb-IIIa is near the epitope defined by the monoclonal antibody PMI-1. This antibody binds to the heavy chain of GPIIb [Shadle et al.,
J. Cell. Biol.,
99:2056-2060 (1984)] and defines a region of GPIIb that is associated with several distinct functional activities. First, PMI-1 inhibits adhesion of washed platelets to collagen. Shadle et al.,
J. Cell. Biol.,
99:2056-2060 (1984). Second, the surface orientation of this region is regulated by divalent cations because millimolar (mM) concentrations of calcium or magnesium suppress expression of the PMI-1 epitope. Ginsberg et al.,
J. Clin. Invest.,
78:1103-1111 (1986). Third, abnormal divalent cation regulation of the conformation of this site is associated with a functional thrombasthenic state. Ginsberg et al.,
J. Clin. Invest.,
78:1103-1111 (1986). Fourth, stimulation of platelets with up to 100 micromolar adenosine diphosphate (ADP) or epinephrine, 1 unit per milliliter thrombin, or 50 micrograms per milliliter calf skin collagen does not substantially increase the binding of PMI-1 antibodies to platelets.
Platelet activation has been reported to produce the appearance of antigenic sites on the platelet surface that are not present in the non-activated platelet, and at least one of such induced sites has been localized to the GPIIb-IIIa receptor complex. Shattil et al,
J. Biol. Chem.
260:11107-11114 (1985); Coller, B. S.,
J. Cell Biol.,
103:451-456 (1986).
Numerous assays based on the release of fragments of blood coagulation enzymes or platelet activation have been developed in attempts to identify thrombotic and pre-thrombotic states. From the platelet perspective, initial efforts were focused on release of platelet specific antigens. Ginsberg, et al.,
In Immunologic Analysis: Recent Progress in Diagnostic Labor. Immunol
., Nakamura et al., Eds. Masson Publishing, USA Inc., 199-209 (1982). Recent studies have focused on the use of monoclonal antibodies selective for activated platelets. Frelinger et al.,
J. Biol. Chem.,
263:12397-12402 (1988); Aiken et al.,
Sem. Thromb. Hemost.,
13:307-316 (1987); Berman et al.,
J. Clin. Invest.,
78:130-137 (1986); Hsu-Lin et al.,
J. Biol. Chem.,
259:9121-9126 (1984); George et al.,
J. Clin. Invest.,
78:340-348 (1986); Shattil et al.,
Blood,
73:150-158 (1989); Shattil et al.,
J. Biol. Chem.,
260:11107-11114 (1985); Adelman et al.,
Blood,
70:1362-1366 (1987).
Each of the above methods has been plagued by unintentional in vitro activation because it is difficult to obtain blood samples in a manner such that the platelet activation events are not triggered during venipuncture, sample handling or the like. This difficulty has severely limited the clinical utility of such assays. It would be desirable, therefore, to develop a method to detect the occurrence of thrombotic events without relying on platelet activation events. The utility of determining the elevation in a plasma glycocalicin, as a potential assay for in vivo platelet activation, has also been reported. Steinberg et al.,
N. Engl. J. Med.,
317:1037-1042 (1987); Coller et al.,
J. Clin. Invest.,
73:794-799 (1984).
It has recently been found that a class of antigenic determinants are expressed when GPIIb-IIIa specifically binds to its ligand. The antigenic determinants are not expressed by either the non-occupied GPIIb-IIIa receptor or the non-bound ligand. One such determinant located on the C-terminus of the GPIIB heavy chain is recognized by the previously described PMI-1 monoclonal antibody. Shadle et al.,
J. Cell Biol.,
99:2056-2060 (1984); Frelinger et al.,
J. Biol. Chem.,
263:12397-12402 (1988).
BRIEF SUMMARY OF THE INVENTION
It has now been found that a portion of the C-terminus of hGPIIb, containing the PMI-1 antigenic determinant, becomes exposed upon receptor-ligand binding and is cleaved by indigenous proteases induced by thrombus formation to form a cleaved fragment. The fragment, referred to as C-terminal hGPIIb fragment, is released into the vascular fluid, and finds its way into other body fluids, such as urine.
Simple activation of platelets with agonists in vitro is not sufficient to release the C-terminal hGPIIb fragment. Treatment with a protease is required. In the thrombus, the platelet receptor is occupied, thereby exposing this region of the molecule, and a variety indigenous proteases, such as plasmin, are generated. Thus, in the present invention, detecting the release of C-terminal hGPIIb fragment by proteases is not subject to the problems of in vitro activation, and can be performed on a routinely obtained plasma or urine specimen from a patient.
The principal utility of the present invention is its ability to identify in vitro thrombotic events ongoing in vivo, and to monitor thrombolysis. Clinical settings include acute thrombotic events such as stroke or coronary thrombosis, or more chronic events such as deep venous thrombosis. In addition, detection of chronic ongoing thrombotic events can serve to identify individuals at risk of developing acute thrombosis.
The present invention is directed to a substantially isolated C-terminal hGPIIb fragment that has a molecular weight of about 3900 daltons, comprising an amino acid residue sequence that corresponds to the carboxy terminal portion of the amino acid residue sequence of hGPIIb shown in SEQ ID NO 3, including the amino acid residue sequence represented by the formula -IHPAHHK-, shown in SEQ ID NO 3 from residue 194 to residue 200. This fragment has the capacity to immunoreact with the monoclonal antibody PMI-1, and preferably has the amino acid residue sequence comprising the amino acid residue sequence shown in SEQ ID NO 3 from about residue 173 to about residue 200.
Also contemplated is a hGPIIb analog comprising a polypeptide of no more than about 200 residues, having an amino acid residue sequence that corresponds to the sequence of hGPIIb shown in SEQ ID NO 3 and includes at least about 7 contiguous amino acid residues from the amino acid residue sequence of hGPIIb shown in SEQ ID NO 3 from residue 173 to

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