Complex prothrombinase

The protein-C pathway is one of the most important anticoagulant mechanisms. It is activated by thrombin. Thrombin binds to a cofactor in the membrane of endothelial cells, thrombomodulin (TM). TM bound thrombin no longer activates clotting factors or platelets but becomes an effective protein C (PC) activator. Activated PC (APC) forms a complex with Protein S, which inactivates FVIIIa and FVa. Hereby generation of Flla by the prothrombinase complex is inhibited (Fig. 9). Thus, the PC-pathway controls thrombin generation in a negative feedback manner. 

Activated on surface of activated platelets by prothrombinase complex (Ca, factors Va and Xa). 

The activation of prothrombin, like that of factor X, occurs on the surface of activated platelets and requires the assembly of a prothrombinase complex, consisting of platelet anionic phospholipids, Ca, factor Va, factor Xa, and prothrombin. 

Thrombin (34 kDa), a serine protease formed by the prothrombinase complex, hydrolyzes the four Arg-Gly bonds between the fibrinopeptides and the a and P portions of the Aa and BP chains of fibrinogen (Figure 51-5A). The release of the fibrinopeptides by thrombin generates fibrin monomer, which has the subunit stmc-... 

Fig. 2. Generation of tenase and prothrombin complexes. PPL represents the anionic phospholipid surface provided by the platelets (platelet phospholipid). Cleavage of prothrombin by the prothrombinase complex results in the formation of thrombin and the release of a small fragment called prothrombin fragment 1.2 (PFI.2).

Tracy P. B Rorhbach M. S Mann K. G. Functional prothrombinase complex assembly on isolated monocytes and lymphocytes. J Biol Chem 1983 258,7264-7. 

Chattopadhyay, A., James, H.I., and Fair, P.S. (1992) Molecular recognition sites on factor Xa which participate in the prothrombinase complex./. Biol. Ghem. 267, 12323-12329. 

The appearance of anionic phospholipids, particularly phosphatidylserine, on the cell siuface activates prothrombinase complex culminating in the formation of thrombin (Bevers et al., 1982 Connor et al., 1989). The assay can be performed with pure coagulation proteins and specific chromogenic substtates to produce a very sensitive test to detect the appearance of phosphatidylserine on ceU siufaces. Nevertheless, it has been shown that changes in the disposition of phosphatidylethanolamine and sphingomyelin may interfere with the ability of phosphatidylserine-containing membranes to activate prothrombinase (Smeets et al., 1996). 

Connor, J., Bucana, C, Fidler, l.J. and Schroit, A.J., 1989, Differentiation-dependent expression ofphosphatidylseiine in mammalian plasma membranes quantitative assessment of outer leaflet lipid by prothrombinase complex formation. Proc. Natl. Acad. ScL, U.S.A. 86 3184-3188. 

Comfurius, P., Smeets, E.F., Willems, G.M., Bevers, E.M., and Zwaal,R.F.A., 1994, Assembly of the prothrombinase complex on the hpid vesicles dependent on the stereochemical configuration of polar headgroup of phosphatidylserine. Biochemistry 33 10319-10324. 

Both pathways depend on the presence of activated thrombocytes, on the surface of which several reactions take place. For example, the prothrombinase complex (left) forms when factors Xa and 11, with the help of Va, bind via Ca "" ions to anionic phospholipids in the thrombocyte membrane. For this to happen, factors 11 and X have to contain the non-proteinogenic amino acid y-carboxygluta-mate (Gla see p. 62), which is formed in the liver by post-translational carboxylation of the factors. The Gla residues are found in groups in special domains that create contacts to the Ca "" ions. Factors Vll and IX are also linked to membrane phospholipids via Gla residues. 

Platelet membrane phosphatidylserine is critical to the formation of the tenase complex since on its surface activated factor VIII (Villa) generates a high-afflnity binding site for activated factor IX (IXa) in the presence of calcium. Subsequently, this complex activates factor X (2, 13). Platelet membrane phosphatidylserine similarly anchors activated factor V (Va), favoring the calcium-dependent binding of activated factor X (Xa). The prothrombinase complex is generated on the surface of the anionic platelet membrane phosphatidylserine when factor Va binds prothrombin. The prothrombinase complex cleaves prothrombin to produce thrombin, which has a multifunctional role (14). 

On surface of platelet membrane phospholipid, phosphatidyl serine forms tenase and prothrombinase complexes... 

Nesheim ME, Kettner C, Shaw E, Mann KG. Cofactor dependence of factor Xa incorporation into the prothrombinase complex. JBiol Chem 1981 256 6537- 6540. 

Sims PJ, Wiedmer T, Esmon CT, Weiss HJ, Shattil SJ. Assembly of the platelet prothrombinase complex is linked to vesicula-tion of the platelet plasma membrane. J Biol Chem 1989 264 17,049-17,057. 

Tracy PB, Eide LL, Mann KG. Human prothrombinase complex assembly and function on isolated peripheral blood cell populations,] Biol Chem 1985 260 21 19-2124. 

Platelet participation in normal hemostasis. The hemostatic plug is the specific response to external vessel lesion and depends on the extent of vessel wall damage, the specific interaction between endothelial cells and activated platelets, release of the contents of platelets intracellular granules in response to activation, the conjoint activity of activated factor Vll and platelet agonists, and the open conditions of blood flow. After activation, platelets also produce the external ization of membrane phosphatidylserine through the flip-flop mechanism that will support the function of the prothrombinase complex ending in thrombin generation and local clot formation. 

It is now widely accepted that atherosclerosis is a chronic inflammatory arterial disease associated with risk factors, platelet, and other blood cells activities and their interactions with subendothelial cells, Activated platelets release active components from citosol and induce the externalization of phosphatidylserine through the flip-flop mechanism (23) that supports the function of the prothrombinase complex ending in thrombin generation,... 

Schematic view of the role of coagulation factor Xa in arterial thrombosis. After endothelial injury, platelets adhere to the subendothelial matrix. The procoagulant activity of the arterial clot can be attributed to the formation of the prothrombinase complex on the platelet surface which cleaves prothrombin and produces thrombin. Thrombin subsequently acts as a strong agonist of further platelet aggregation.

New small molecule FXa inhibitors currently in development are able to enter the clot/prothrombinase complex and inhibit free and bound factor Xa regarded as the key enzyme in ACS. Although direct FXa inhibitors do not inhibit platelet aggregation, they abolish platelet-dependent thrombus formation in canine coronary thrombosis. Thus, direct inhibition of FXa may have higher efficacy and better risk/benefit profile than existing antithrombotic therapies in the treatment and prevention of ACS. 

An important step in the blood coagulation pathway is the formation of the prothrombinase complex. The latter is a mixture of factor V, factor Xa, Ca2+, and phospholipid. In this case, a phospholipid mixture with a net negative charge will allow the prothrombinase complex to form. This active enzyme is important in cleaving prothrombin to yield thrombin. The most active phospholipid mixture for in vitro studies has proven to be phosphatidylserine-phosphatidylcholine. Subsequently the hypothesis has developed that phosphatidylserine is key to the formation of prothrombinase. 

The composition of the cotton threaded thrombus shows a composition of fibrin together with tightly aggregated and distorted erythrocytes, thus being in accordance with human deep vein thrombosis structure. Non-occlusive thrombus formation has been successfully inhibited by heparins, prothrombinase complex inhibitors and thrombin inhibitors (Hollenbach et al. 1994,1995). 

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