Anticoagulation agent and uses thereof

The present invention relates to the field of haematology and particularly the sub-specialty of haemostasis. More specifically, the present invention relates to 1) agents that inhibit coagulation in mammalian blood, and uses for these agents in the treatment and prevention of diseases such as stroke, myocardial infarction, and deep vein thrombosis and 2) probes that allow diagnosis and identification of activated platelets in clinical settings such as thrombosis, thrombotic emboli as well as unstable plaques.

The ability of the body to control the flow of blood following vascular injury is paramount to continued survival. The process of blood clotting and then the subsequent dissolution of the clot, following repair of the injured tissue, is termed hemostasis. Hemostasis is composed of a number of events that occur in a set order following the loss of vascular integrity:

The initial phase of the process is vascular constriction. This limits the flow of blood to the area of injury. Next, platelets become activated by thrombin and aggregate at the site of injury, forming a temporary, loose platelet plug. The protein fibrinogen is primarily responsible for stimulating platelet clumping. Platelets clump by binding to collagen that becomes exposed following rupture of the endothelial lining of vessels. Upon activation, platelets release adenosine-5′-diphosphate, ADP and TXA2 (which activate additional platelets), serotonin, phospholipids, lipoproteins, and other proteins important for the coagulation cascade. In addition to induced secretion, activated platelets change their shape to accommodate the formation of the plug.

To insure stability of the initially loose platelet plug, a fibrin mesh (also called the dot) forms and entraps the plug. Finally, the clot must be dissolved in order for normal blood flow to resume following tissue repair. The dissolution of the clot occurs through the action of plasmin.

Two pathways lead to the formation of a fibrin clot: the intrinsic and extrinsic pathway. Although they are initiated by distinct mechanisms, the two converge on a common pathway that leads to clot formation. The formation of a red thrombus or a clot in response to an abnormal vessel wall in the absence of tissue injury is the result of the intrinsic pathway. Fibrin clot formation in response to tissue injury is the result of the extrinsic pathway. Both pathways are complex and involve numerous different proteins termed clotting factors

Platelet Activation and Von Willebrand Factor (vWF).

In order for hemostasis to occur, platelets must adhere to exposed collagen, release the contents of their granules, and aggregate. The adhesion of platelets to the collagen exposed on endothelial cell surfaces is mediated by von Willebrand factor (vWF). The function of vWF is to act as a bridge between a specific glycoprotein on the surface of platelets (GPlb/IX) and collagen fibrils. In addition to its role as a bridge between platelets and exposed collagen on endothelial surfaces, vWF binds to and stabilizes coagulation factor VIII. Binding of factor VIII by vWF is required for normal survival of factor VIII in the circulation.

Von Willebrand factor is a complex multimeric glycoprotein that is produced by and stored in the platelets. It is also synthesized by megakaryocytes and found associated with subendothelial connective tissue. The initial activation of platelets is induced by thrombin binding to specific receptors on the surface of platelets, thereby initiating a signal transduction cascade. The thrombin receptor is coupled to a G-protein that, in turn, activates phospholipase C-γ (PLC-γ). PLC-γ hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP2) leading to the formation of inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 induces the release of intracellular Ca2+ stores, and DAG activates protein kinase C (PKC).

The collagen to which platelets adhere as well as the release of intracellular Ca2+ leads to the activation of phospholipase A2 (PLA2), which then hydrolyzes membrane phospholipids, leading to liberation of arachidonic acid. The arachidonic acid release leads to an increase in the production and subsequent release of thromboxane A2 (TXA2). This is another platelet activator that functions through the PLC-γ pathway. Another enzyme activated by the released intracellular Ca2+ stores is myosin light chain kinase (MLCK). Activated MLCK phosphorylates the light chain of myosin which then interacts with actin, resulting in altered platelet morphology and motility.

One of the many effects of PKC is the phosphorylation and activation of a specific 47,000-Dalton platelet protein. This activated protein induces the release of platelet granule contents; one of which is ADP. ADP further stimulates platelets increasing the overall activation cascade; it also modifies the platelet membrane in such a way as to allow fibrinogen to adhere to the platelet surface, resulting in fibrinogen-induced platelet aggregation.

Activation of platelets is required for their consequent aggregation to a platelet plug. However, equally significant is the role of activated platelet surface phospholipids in the activation of the coagulation cascade.

The intrinsic clotting cascade is initiated when contact is made between blood and exposed endothelial cell surfaces. The extrinsic and intrinsic pathways converge at the point where factor X is activated to factor Xa. Factor Xa has a role in the further activation of factor VII to VIIa. Active factor Xa also hydrolyzes and activates prothrombin to thrombin. Thrombin can then activate factors XI, VIII and V furthering the cascade. Ultimately the role of thrombin is to convert fibrinogen to fibrin and to activate factor XIII to XIIIa. Factor XIIIa (also termed transglutaminase) cross-links fibrin polymers solidifying the clot.

The intrinsic pathway requires the clotting factors VIII, IX, X, XI, and XII. Also required are the proteins prekallikrein and high-molecular-weight kininogen, as well as calcium ions and phospholipids secreted from platelets. Each of these pathway constituents leads to the conversion of factor X (inactive) to factor Xa (active). Initiation of the intrinsic pathway occurs when prekallikrein, high-molecular-weight kininogen, factor XI and factor XII are exposed to a negatively charged surface. This is termed the contact phase. Exposure of collagen to a vessel surface is the primary stimulus for the contact phase.

The assemblage of contact phase components results in conversion of prekallikrein to kallikrein, which in turn activates factor XII to factor XIIa. Factor XIIa can then hydrolyze more prekallikrein to kallikrein, establishing a reciprocal activation cascade. Factor XIIa also activates factor XI to factor XIa and leads to the release of bradykinin, a potent vasodilator, from high-molecular-weight kininogen.

In the presence of Ca2+, factor XIa activates factor IX to factor IXa. Factor IX is a proenzyme that contains vitamin K-dependent γ-carboxyglutamate (gla) residues, whose serine protease activity is activated following Ca2+ binding to these gla residues. Several of the serine proteases of the cascade (II, VII, IX, and X) are gla-containing proenzymes. Active factor IXa cleaves factor X at an internal arg-ile bond leading to its activation to factor Xa.

The activation of factor Xa requires assemblage of the tenase complex (Ca2+ and factors VIIIa, IXa and X) on the surface of activated platelets. One of the responses of platelets to activation is the presentation of phosphatidylserine and phosphatidylinositol on their surfaces. The exposure of these phospholipids allows the tenase complex to form. The role of factor VIII in this process is to act as a receptor, in the form of factor VIIIa, for factors IXa and X. Factor VIIIa is termed a cofactor in the clotting cascade. The activation of factor VIII to factor VIIIa (the actual receptor) occurs in the presence of minute quantities of thrombin. As the concentration of thrombin increases, factor VIIIa is ultimately cleaved by thrombin and inactivated. This dual action of thrombin, upon factor VIII, acts to limit the extent of tenase complex formation and thus the extent of the coagulation cascade.

As discussed supra activated factor Xa is the site at which the intrinsic and extrinsic coagulation cascades converge. The extrinsic pathway is initiated at the site of injury in response to the release of tissue factor (factor III). Tissue factor is a cofactor in the factor VIIa-catalyzed activation of factor X. Factor VIIa, a gla residue containing serine protease, cleaves factor X to factor Xa in a manner identical to that of factor IXa of the intrinsic pathway. The activation of factor VII occurs through the action of thrombin or factor Xa. The ability of factor Xa to activate factor VII creates a link between the intrinsic and extrinsic pathways. An additional link between the two pathways exists through the ability of tissue factor and factor VIIa to activate factor IX. While there is some uncertainty it appears the formation of complex between factor VIIa and tissue factor is believed to be a principal step in the overall clotting cascade. A major mechanism for the inhibition of the extrinsic pathway occurs at the tissue factor-factor VIIa-Ca2+-Xa complex. The protein, lipoprotein-associated coagulation inhibitor, LACI specifically binds to this complex. LACI is also referred to as extrinsic pathway inhibitor, EPI or tissue factor pathway inhibitor, TFPI and was formerly named anticonvertin. LACI is composed of 3 tandem protease inhibitor domains. Domain 1 binds to factor Xa and domain 2 binds to factor VIIa only in the presence of factor Xa

The common point in both extrinsic and intrinsic pathways is the activation of factor X to factor Xa. Factor Xa activates prothrombin (factor II) to thrombin (factor IIa). Thrombin, in turn, converts fibrinogen to fibrin. The activation of thrombin occurs on the surface of activated platelets and requires formation of a prothrombinase complex. This complex is composed of the platelet phospholipids, phosphatidylinositol and phosphatidylserine, Ca2+, factors Va and Xa, and prothrombin. Factor V is a cofactor in the formation of the prothrombinase complex, similar to the role of factor VIII in tenase complex formation. Like factor VIII activation, factor V is activated to factor Va by means of minute amounts and is inactivated by increased levels of thrombin. Factor Va binds to specific receptors on the surfaces of activated platelets and forms a complex with prothrombin and factor Xa.

Prothrombin is a 72,000-Dalton, single-chain protein containing ten gla residues in its N-terminal region. Within the prothrombinase complex, prothrombin is cleaved at 2 sites by factor Xa. This cleavage generates a 2-chain active thrombin molecule containing an A and a B chain which are held together by a single disulfide bond.

In addition to its role in activation of fibrin clot formation, thrombin plays an important regulatory role in coagulation. Thrombin combines with thrombomodulin present on endothelial cell surfaces forming a complex that converts protein C to protein Ca. The cofactor protein S and protein Ca degrade factors Va and VIIIa, thereby limiting the activity of these two factors in the coagulation cascade.