Method for sustaining enos activity

a technology of enos activity and kit, which is applied in the field of methods and kits for sustaining enos activity, can solve the problems of ischemic necrosis of the tissue supplied by the artery, disease state, morbidity and mortality, etc., and achieve the effects of inhibiting the activity of syk kinase, inhibiting platelet aggregation and clot retraction, and promoting fibrinolysis

Inactive Publication Date: 2006-06-15
SARKAR SIBAJI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Intravascular thrombosis is one of the most frequent pathological events and a major cause of morbidity and mortality.
While clotting as a result of an injury to a blood vessel is a critical physiological process for mammals such as man, inappropriate clotting can also lead to disease states.
Arterial thrombosis may result in ischemic necrosis of the tissue supplied by the artery.
When the thrombosis occurs in a coronary artery, a myocardial infarction or heart attack can result.
A thrombosis occurring in a vein may cause tissues drained by the vein to become edematous and inflamed.
Thrombosis of a deep vein may be complicated by a pulmonary embolism.
However, while activity has focused on inducing nitric oxide synthase activity, it has not been previously known how to regulate constitutive endothelial nitric oxide synthase (eNOS) activity.
Anti-thrombotic agents can block or inhibit thrombus formation, as discussed above; however, they are not very effective in dissolving a pre-formed thrombus or to help in fibrinolysis.
Thus, terminal thrombus formation may cause myocardial infarction and / or ischemic chest pain.
However, fibrinolytic agents typically have problems because of the inhibitory effect of platelets on clot lysis.
However, platelet PAI-1 exists predominantly in a latent or inactive form, suggesting its effect on fibrinolysis to be rather limited.

Method used

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Examples

Experimental program
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Effect test

example 1

Inhibition of Clot Retraction

[0108] To determine the effect of the syk kinase inhibitor piceatannol and the calpain inhibitor calpeptin, the following experiments were carried out. Platelets (2×108 platelets / ml) were incubated with either piceatannol at a final concentration of 40 μg / ml, calpeptin at a final concentration of 200 μg / ml, or vehicle DMSO for 30 minutes in the dark. To each sample, fibrinogen was added to a final concentration of 300 nM, 1 mM Ca2+, and 2 mM Mg2+, 10 minutes prior to the addition of 0.5 unit / ml of thrombin. The clots were incubated for 30 minutes at 37° C. and then transferred to ice before taking photographs. Tubes: 1, vehicle control treated sample without the addition of thrombin; 2, vehicle control treated sample; 3, piceatannol treated sample; 4, calpeptin treated sample.

[0109] Piceatannol at 20 ug / ml and 40 ug / ml inhibited clot retraction where as PP2 at 10 μM did not. As shown in FIG. 1, no clot retraction was observed in tube 1 when thrombin wa...

example 2

Nitric Oxide Production

[0111]FIG. 2 shows the effect of calpeptin on NO production. Platelets at 2×108 / ml were pre-incubated with either calpeptin at final concentration 200 ug / ml or vehicle DMSO for 30 minutes in the dark. To a final volume of 0.5 ml, fibrinogen was added to a final concentration of 1 mg / ml, 1 mM Ca2+, and 2 mM Mg2+, and GPRP to a final concentration of 20 μM. The tube was placed in the NO-501 monitor to measure NO production and the platelets are stirred with a magnetic stirrer. TRAP (Thrombin Receptor Agonist Peptide) at a final concentration of 20 μM was added to start the reaction. The red line shows the platelet NO production and the blue line platelet aggregation. The readings were taken for 300 seconds (5 minutes).

[0112] Calpeptin at 200 μg / ml increased and sustained NO production. In vitro experiments have shown that increased NO production inhibited platelet recruitment. FIG. 2 showed that protease calpain inhibitor Calpeptin increased and sustained NO p...

example 3

Clot lysis

[0116] Anti-thrombotic agents can block or inhibit thrombus formation but they are not much effective on pre-formed thrombus to dissolve them and / or to help in fibrinolysis at t-PA or u-PA. Thus terminally full thrombus formation may cause myocardial-infarction (heart attack) and / or ischemic chest pain. The treatment for total blockage by thrombus formation is so far either angio-baloon-plasty and / or by-pass surgery. Fibrinolytic agents do not usually work as plasma plasminogen could not be converted to plasmin as active platelets at the site of thrombus formation secrete molecules such as PAI which inhibit proteolytic processing of plasminogen to generate active plasmin.

[0117] To assess the effect of piceatannol and calpeptin on the fibrinolytic effect of t-PA and plasminogen in the presence of platelets, we designed a two step procedure by modifying the fibrinolytic assay as described by Fay et al. (Blood, 83, 351-356, 1994).

[0118] First, we developed a retracted clot...

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Abstract

The present invention is directed to methods for sustaining eNOS activity to inhibit platelet aggregation, clot retraction, and enhance fibrinolysis. One embodiment of the invention provides methods of treating thrombosis by inhibiting the activity of the syk kinase. Another embodiment provides assays for the discovery of improved compounds to treat thrombosis, by screening for compounds which sustain eNOS activity. Yet another embodiment provides assays for the discovery of improved compounds to treat thrombosis, by identifying inhibitors of calpain and IIbIIIa by screening for compounds which act through calpain or IIbIIIa to sustain eNOS activity. Yet another embodiment provides for enhancing fibrinolysis, by inhibiting the activity of the syk kinase or calpain.

Description

CROSS-REFERENCE TO RELATED APPLICATION [0001] This application claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Patent Application Ser. No. 60 / 431,633, filed Dec. 6, 2002.FIELD OF THE INVENTION [0002] The present application is directed to methods and kits for sustaining eNOS activity. These methods and kits can be used to treat thrombosis by inhibiting platelet aggregation and clot retraction, and enhancing fibrinolysis. BACKGROUND OF THE INVENTION [0003] Intravascular thrombosis is one of the most frequent pathological events and a major cause of morbidity and mortality. Critical steps in the development of acute coronary syndromes are the disruption, rupture, or erosion of artherosclerotic plaques with the formation of either partially or completely occlusive thrombus. Factors that stimulate thrombosis include vascular damage, stimulation of platelets, and activation of the coagulation cascade. Platelet adhesion to the exposed subendothelial surfaces of injured blood...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/54A61K31/05A61K31/27
CPCA61K31/05A61K31/27G01N2333/90254C12Q1/26G01N33/86
Inventor SARKAR, SIBAJIFREEDMAN, JANEVARGHESE, SONIA
Owner SARKAR SIBAJI
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