Phospholipids thrombin

Figure 51-1. The pathways of blood coagulation. The intrinsic and extrinsic pathways are indicated. The events depicted below factor Xa are designated the final common pathway, culminating in the formation of cross-linked fibrin. New observations (dotted arrow) include the finding that complexes of tissue factor and factor Vila activate not only factor X (in the classic extrinsic pathway) but also factor IX in the intrinsic pathway, in addition, thrombin and factor Xa feedback-activate at the two sites indicated (dashed arrows). (PK, prekallikrein HK, HMW kininogen PL, phospholipids.) (Reproduced, with permission, from Roberts HR, Lozier JN New perspectives on the coagulation cascade. Hosp Pract [Off Ed] 1992Jan 27 97.)...

Figure 51-2. Diagrammatic representation (not to scale) of the binding of factors Va, Xa, Ca +, and prothrombin to the plasma membrane of the activated platelet. The sites of cleavage of prothrombin by factor Xa are indicated by two arrows. The part of prothrombin destined to form thrombin is labeled prethrombin.The Ca " is bound to anionic phospholipids of the plasma membrane of the activated platelet.

Activated partial thromboplastin time aPTT is performed by adding calcium phospholipids and kaolin to citrated blood and measures the time required for a fibrin clot to form. In this manner, aPTT measures the activity of intrinsic and common pathways. Prolongation of aPTT may be due to a deficiency or inhibitor for factors II, V, VIII, IX, X, XI, and XII. It also may be due to heparin, direct thrombin inhibitors, vitamin K deficiency, liver disease, or lupus anticoagulant. 

G21. DeGroot, P. G Gonsalves, M. D., Loesberg, C., Van Buul-Wortelboer, M. F., Van Aken, W. G and Van Mourik, J. A., Thrombin-induced release of von Willebrand factor from endothelial cells is mediated by phospholipid methylation. J. Biol. Chem. 259, 13329-13333 (1984). 

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).

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). 

Normally, thrombin is present in the blood as an inactive proenzyme (see p. 270). Prothrombin is activated in two different ways, both of which represent cascades of enzymatic reactions in which inactive proenzymes (zymogens, symbol circle) are proteolytically converted into active proteinases (symbol sector of a circle). The proteinases activate the next proenzyme in turn, and so on. Several steps in the cascade require additional protein factors (factors 111, Va and Villa) as well as anionic phospholipids (PL see below) and Ca "" ions. Both pathways are activated by injuries to the vessel wall. 

Disruption of the endothelial surface of blood vessels expose collagen fibers and connective tissue. These provide surfaces that promote platelet adherence, platelet release reaction, and subsequent platelet aggregation. Substances liberated from the platelets stimulate further platelet aggregation, eg, adenosine diphosphate maintain vasoconstriction, eg, serotonin and participate in blood coagulation, eg, platelet Factors III and IV. In addition, the release reaction modifies platelet membranes in a manner that renders phospholipid available for coagulation. The thrombin [9002-04-4] elaborated by the coagulation mechanism is a potent agent in the induction of the platelet release reaction. 

Factor V. High in sialic acid content, Factor V is a large asymmetric single-chain glycoprotein that becomes an active participant in the coagulation cascade when it is converted to its active form by a-thrombin. Approximately 25% of human Factor V is found in the whole blood associated with platelets. Factor V is an essential cofactor along with Factor Xa plus phospholipid plus Ca2+ in the conversion of prothrombin to thrombin. 

The most obvious effect of a deficiency in vitamin K in animals is delayed blood clotting, which has been traced to a decrease in the activity of prothrombin and of clotting factors VII, IX, and X (Chapter 12, Fig. 12-17). Prothrombin formed by the liver in the absence of vitamin K lacks the ability to chelate calcium ions essential for the binding of prothrombin to phospholipids and to its activation to thrombin. The structural differences between this abnormal protein and the normal prothrombin have been pinpointed at the N terminus of the 560 residue glycoprotein.e f Tryptic peptides from the N termini differed in electrophoretic mobility. As detailed in Chapter 12, ten residues within the first 33, which were identified as glutamate residues by the sequence analysis on normal prothrombin, are actually y-carboxyglutamate (Gla). The same amino acid is present near the N termini of clotting factors VII, IX, and X. 

The blood coagulation cascade. Each of the curved red arrows represents a proteolytic reaction, in which a protein is cleaved at one or more specific sites. With the exception of fibrinogen, the substrate in each reaction is an inactive zymogen except for fibrin, each product is an active protease that proceeds to cleave another member in the series. Many of the steps also depend on interactions of the proteins with Ca2+ ions and phospholipids. The cascade starts when factor XII and prekallikrein come into contact with materials that are released or exposed in injured tissue. (The exact nature of these materials is still not fully clear.) When thrombin cleaves fibrinogen at several points, the trimmed protein (fibrin) polymerizes to form a clot. 

Arachidonic acid is not present in significant amounts in tissues as the free acid but is stored as a fatty acid at the sn-2 position of phospholipids. Prostaglandin biosynthesis is initiated by the interaction of a stimulus with the cell surface. Depending on the cell type, the stimulus can take the form of a hormone, such as angiotensin II or antidiuretic hormone, or a protease such as thrombin (involved in blood clotting), or both hormone and protease. These agents bind to a specific receptor that activates a phospholipase A2 that specifically releases the arachidonic acid from a phospholipid such as phosphatidylcholine. The release of arachidonic acid by phospholipase A2 is believed to be the rate-limiting step for the biosynthesis of eicosanoids. 

Antibodies directed against (32 -GPI further enrich the protein on the cell surface but these promote a p38 Map-kinase signalling cascade which results in increased expression of tissue factor (TF) and reduced expression of thrombomodulin on the surface of cells. TF is a major initiator of coagulation and increased levels of TF expression have been measured on endothelial cells treated with anti-phospholipid antibodies and on monocytes both ex vivo and in vivo (Yasuda et al., 2005 Lopez-lira et al., 2005, Lopez-Pedrera et al., 2006). Thrombomodulin is a potent anti-coagulant protein which limits activation of thrombin, so the net result of circulating anti-phospholipid antibodies is to usurp the anti-coagulative, protective mechanism and initiate a pro-coagulation cascade. 

Dachary-Prigent J, Toti F, Satta N, Pasquet JM, Uzan A, Freyssinet JM. Physiopatho-logical significance of catalytic phospholipids in the generation of thrombin. Semin Thromb Hemost 1996 22 157-164. 

The negatively charged phospholipid phosphatidylserine is asymmetrically distributed in mammalian cell membranes, primarily on the inner leaflet. Upon exposure to collagen or thrombin, the distribution of phospholipids changes with increasing phosphatidylserine in the external membrane leaf (I). The increased expression of phosphatidylserine on the outer leaflet of the membrane creates a procoagulant surface on which several steps of the coagulation cascade take place. 

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. 

Thrombin (MW 39,000) is a proteolytic enzyme of the serine protease group. It is derived from prothrombin, a circulating plasma protein, through the proteolytic action of a complex consisting of the proteolytic enzyme factor X (or factor Xa), another protein called factor V (accelerator protein), calcium, and phospholipid. Factor V has recently been identified as the plasma copper protein ceruloplasmin or a similar protein (see Chapter 6). 

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