Prothrombinase

In addition to the AT-dependent agents discussed above, various direct Xa inhibitors (e.g., tick anticoagulant peptide, antistatin, DX-9065a) are undergoing clinical testing. Unlike fondaparinux, these drugs also inhibit surface-bound Xa within 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... 

Formation of prothrombinase It can be formed by extrinsic pathway and intrinsic pathway. 

Extrinsic pathway This pathway has fewer steps than the intrinsic pathway and occurs rapidly, within a matter of seconds if the trauma is severe. It is called the extrinsic pathway because a protein tissue factor, also called thromboplastin or coagulation factor III, takes into the blood stream from outside and initiates the formation of prothrombinase. Tissue factor is released from the surface of the damaged cells. It activates factor VII. Factor VII combines with factor X, activating it. Factor X in the presence of Ca combines with factor V to give active enzyme prothrombinase. 

Once prothrombinase has been formed, the common pathway is followed. In stage 2, prothrombinase and calcium catalyze the conversion of prothrombin to thrombin. In stage 3, thrombin, in the presence of calcium converts soluble fibrinogen to insoluble fibrin threads. 

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

Thrombin activates platelets, converts fibrinogen to fibrin, activates factor XIII, which stabilizes fibrin, and activates factors V and VIE, which accelerate the generation of prothrombinase. Therefore, the inhibition of thrombin is essential in preventing and treating thromboembolic disorders. 

As of yet, there is no consensus as to which marker is more sensitive or specific to detect EMP. In addition, there are two main quantitative methodologies for detecting EMPs currently in use flow cytometry (FQ and a combination of AV capture (AVQ and prothrombinase assay (PTA) with ELISA [5, 6]. Table 1 summarizes the main features of the clinical assays most commonly used so far. 

Hamilton KK, Hattori R, Esmon CT, Sims PJ. Complement proteins C5b-9 induce vesiculation of the endothelial plasma membrane and expose catalytic surface for assembly of the prothrombinase enzyme complex. J Biol Chem 1990 265 3809-3814. 

Prothrombin has been shown to be efficiently activated on the cell surface of monocytes and lymphocytes (65), As with platelets, the prothrombinase activity on monocytes is increased with activated monocytes as compared to the nonactivated cells (66). 

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