Antiplatelet agent

Details Antiplatelet agent Antiplatelet agent

1999-12-07

2002-12-03

Carlson, Karen Cochrane (Department: 1653)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S008100, C514S012200, C530S324000, C530S345000, C530S380000, C530S383000, C530S395000, C435S069600

Reexamination Certificate

active

06489290

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention is directed to anti-thrombotic therapy using novel anti-thrombotic agents. In particular, the anti-thrombotic agents are S-nitrosated polypeptides which bind to platelet receptor glycoprotein GPIb/IX.
2. Review of Related Art
von Willebrand Factor and Platelet Function
During the past several years significant progress has been made in understanding the molecular aspects of platelet function with regard to both their role in normal hemostasis and the development of pathological vascular occlusion. The first event in normal primary hemostasis or development of arterial thrombosis is the binding (adhesion) of platelets to the subendothelium at sites of vascular injury. This first step occurs by binding of von Willebrand factor (vWF) to the platelet receptor glycoprotein Ib/IX (GPIb/IX) following its binding to components of exposed subendothelium. Thus, vWF acts as a “bridging” molecule between platelets and the vessel wall. As a consequence of vWF binding to GPIb, glycoprotein IIb/IIIa (GPIIb/IIIa) is activated through a complex signaling pathway leading to platelet aggregation mediated by fibrinogen or, under conditions of high shear stress, by vWF itself (Ruggeri, et al., 1982,
Proc. Natl. Acad. Sci. USA,
79:6038-6041; Ruggeri, et al., 1983,
J. Clin. Invest.,
72:1-12; Ikeda, et al., 1991,
J. Clin. Invest.,
87:1234-1240). Furthermore, the binding of vWF to GPIb can mediate events that are associated with the effects of agonists like ADP and thrombin, such as activation of GPIIb/IIIa and support of platelet aggregation. This indicates that vWF serves as more than the “glue” that mediates platelet adhesion to the vessel wall but also induces and modulates other later steps of hemostasis and thrombogenesis.
vWF is a polymeric glycoprotein that circulates in plasma as a series of multimers with molecular weights ranging from 0.25×10
6
daltons to 20×10
6
daltons. In addition to its role in platelet adhesion, it carries and stabilizes factor VIII in the circulation (Sadler, 1991,
J Biol. Chem.,
266:22777-22780). The vWF gene, located on chromosome 12, spans 178 kb and is interrupted by 51 introns (Ginsburg, et al., 1985,
Science,
228:1401; Mancuso, et al., 1989,
J. Biol. Chem.,
264:19514-19527.) vWF is synthesized from an 8.7 kb mRNA and is expressed in endothelial cells and megakaryocytes. Synthesis of vWF is a complex multistep process that results in the generation of a precursor protein, pre-pro-vWF (Meyer, et al., 1993,
Thromb. Haemost.,
70:99-104).
This large molecule comprises a 22 amino acid (aa) signal peptide, as well as prov WF, which consists of a 741 aa propeptide and a 2050 aa mature subunit. These 250 kDa subunits assemble into multimers of up to 100 subunits (Wagner, 1990,
Annu. Rev. Cell Biol.,
6:217-246). After dimerization by disulfide bonding at carboxyterminal domains in the endoplasmic reticulum, further multimerization takes place in the Golgi or post-Golgi compartments through disulfide linkages at amino-terminal domains.
In the blood vessel, vWF is constitutively secreted by endothelial cells. vWF is also stored within intracellular granules in both endothelial cells (Weibel-Palade bodies) and platelets (&agr;-granules). These specialized granules release vWF in response to a variety of stimuli including vascular damage. The vWF stored within these granules contains larger multimers than those which are constitutively secreted by endothelial cells. These high-molecular-weight (HMW) multimers are more effective in platelet binding than smaller sized multimers (Gralnick, et al., 1981,
Blood,
58:397-397; Federici, et al., 1989,
British Journal of Hematology,
73:93-99); therefore, rapid release of stored vWF into the circulation may be particularly useful in the setting of vessel injury.
The pro vWF consists of four types of repeated domains (A to D) and has two disulfide loops: one is located between cys 509 and 695 in the A1 domain and the other between cys 923 and 1109 in the A3 domain (Meyer, et al., 1993,
Thromb. Haemost.,
70:99-104). Progress has been made in identifying specific regions of the vWF subunit that are important for function. The A1 domain contains binding sites for GPIb, sulfatides, and heparin. Using proteolytic or recombinant fragments of vWF, the binding domain for GPIb has been located within the T116 fragments (aa 449-728) which overlaps the A1 loop (Fujimura, et al., 1986,
J. Biol. Chem.,
261:381-385; Cruz, et al., 1993,
J. Biol. Chem.,
268:21238-21245; Sugimoto, et al., 1991,
Biochemistry,
30:5202-5209; Gralnick, et al., 1992,
Proc. Natl. Acad. Sci. USA,
89:7880-4; Azuma, et al., 1991, J. Biol. Chem., 266:12342-12347; Pietu, et al., 1989,
Biochem. Biophys. Res. Commun.,
164:1339-1347; Andrews, et al., 1989,
Biochemistry,
28:8326-8336).
Under its native conformation, human vWF does not spontaneously interact with GPIb. The exposure of the GPI-binding site of vWF can be regulated by a series of physiological or non-physiological events. Most of these events appear to modify the structure and/or the conformation of the A1 region. The binding of vWF to the subendothelium, which spontaneously occurs via the T116 sequence (Denis, et al., 1993,
Arterioscler. Thromb.,
13:398-406), is responsible for the subsequent exposure of the GPIb-binding site of vWF (Sakariassen, et al., 1979,
Nature,
279:635-638). Similarly, collagen and heparin bind to vWF via sequences in the A1 loop (Mohri, et al., 1989,
J. Biol. Chem.,
264:17361-17367) and modulate its interaction with GPIb. Binding of vWF to collagen promotes its interaction with GPIb while binding to heparin inhibits this interaction (Fressinaud, et al., 1988,
J. Lab. Clin. Med.,
112(1):58-67; Sobel, et al., 1991,
J. Clin. Invest.,
87:1787-1793; Savage, et al., 1992,
J. Biol. Chem.,
267(16):11300-11306).
The interaction of vWF with non-physiologic modulators of its binding to GPIb also involves sequences close to or within the A1 loop. The immobilization of vWF on a plastic surface, for example, leads to platelet adhesion via GPIb (Berndt, et al., 1992,
Biochemistry,
31:11144-11151). The interaction of vWF with ristocetin involves 474-488 and 692-708 sequences flanking the A1 loop, whereas botrocetin binds to four sequences within this loop (514-542, 539-553, 569-583 and 629-643) (Sugimoto, et al., 1991,
J. Biol. Chem.,
266:18172-18178; Ginsburg, et al., 1993,
Thromb. Haemost.,
69:177—184). The inhibition of vWF binding to GPIb by polyanionic compounds like aurin tricarboxylic acid (ATA) involves positively charged sequences of the A1 loop (Girma, et al.,
Thromb. Haemost.,
68:707-13, 1992). Finally, the GPIb-binding site can be achieved by the removal of the sialic acid residues from the carbohydrate side chains of vWF (Gralnick, et al., 1985,
J. Clin. Invest.,
75:19-25). Since 9 of the 22 carbohydrate chains of vWF are within the T116 fragment but outside the A1 loop, the net local decrease of the negative charges may be responsible for the exposure of the GPIb-binding site.
Studies of patients with von Willebrand disease (vWD) have confirmed the role of the conformation of the A1 domain in the regulation of vWF binding to GPIb. vWF from patients with type 2B vWD is characterized by an increased capacity to bind to platelet GPIb. Mutations of this type have been identified within the 505-698 aa residues (Ginsburg, et al.,
Thromb. Haemost.,
69:177-84, 1993). The expression of recombinant, mutated vWF has confirmed the direct role of these mutations in the increased affinity of vWF for GPIb (Randi, et al., 1992,
J. Biol. Chem.,
267:21187-21192; Inbal, et al., 1993,
Thromb. Haemost.,
70:1058-1062; Cooney, et al., 1992,
Proc. Natl. Acad. Sci USA,
89:2869-2872; Kroner, et al., 1992,
Blood,
79:2048-2055).
Matsushita, et al. (1995,
J. Biol. Chem.,
270:13406-13414) recently performed charged-to-alanine mutagenesis of the vWF A1 domain to examine the roles of specific charged residues in the interaction of vWF with platelet GPIb. By this approach, amino aci

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