Acyl-thrombin

Of special interest would be the cellular reaction of acyl-thrombin. By incubation of platelet suspCwlun with uaizoyl unuinbiii, me ploieici reactivity was diminished. The same phenomenon could be observed in platelet suspensions whets small amounts of thrombin were added slowly [25]. Further studies on nonhemosiaric reactions of acyl-thrombin nave not yet been... 

In vivo, the infusion of small amounts of thrombin (<200 NIH-U/kg/h) causes defibrinogenation. Because of the slow transformation of fibrinogen, the fibrin monomers formed are eliminated via reactive fibrinolysis and RES without precipitation of fibrin. Upon infusion of higher amounts of thrombin, these clearance mechanisms fail to offset the thrombin action, which then leads to the formation of microthrombi in the peripheral circulation. Correspondingly, the bolus injection of 1500 NIH-U thrombin/kg caused death of the experimental animals. However, a bolus injection of benzoyl-thrombin at a dose corresponding to the amount of 1500 NIH-U/kg caused defibrinogenation only. Therefore, bolus application of acyl-thrombin may initiate an infusion regimen [26]. 

The 6-chloromethyl substituent (series 5 and 6) is required for the inactivation of a-chymotrypsin. Nevertheless, there is only a transient inactivation of HLE and thrombin through the formation of a stable acyl-enzyme in spite of the presence of this group as demonstrated by the spontaneous or hydroxylamine-accelerated reactivation of the treated enzymes (Scheme 11.3, pathway b).21 HLE is specifically inhibited when such an alkylating function is absent (series 7), always through the formation of a transient acyl-enzyme (Table 11.2). 

Such an intermediate is known to be formed in reactions catalyzed by trypsin, chymotrypsin, thrombin, other enzymes of the blood-clotting cascade (except angiotensinconverting enzyme, which is an aspartic protease). An acyl-serine intermediate is also formed in the acetylcholinesterase reaction. The active site serine of this enzyme and the serine proteases can be alkylated by diisopropyl-fluorophosphate. See also Active Site Titration... 

The mechanism of action of anticholinesterases is to form a stable covalent complex with the Achase enzyme. Achase is one of several enzymes known as serine esterases. Other examples include the intestinal enzymes trypsin and chymotrypsin as well as the blood clotting agent thrombin. During the course of the catalysis the alcohol -OH of a serine side chain in the active site of the enzyme forms an ester complex, called the acyl-enzyme, with the substrate. So, acetylcholine will go through similar chemical reactions with Achase. 

In the acylation step a nucleophilic group on one of the amino-acid side chains at the active site behaves as the nucleophile. As we have seen in Section 25-9B, the nucleophile of carboxypeptidase is the free carboxyl group of glutamic acid 270. In several other enzymes (chymotrypsin, subtilisin, trypsin, elastase, thrombin, acetylcholinesterase), it is the hydroxyl group of a serine residue ... 

Veinberg G, Vorona M, Shestakova I, Kanepe I, Lukevics E (2003) Some of the more notable advances concern the development of mechanism-based serine protease inhibitors of elastase, cytomegalovirus protease, thrombin, prostate specific antigen, and cell metastasis and as inhibitors of acyl-CoA cholesterol acyl transferase. Curr Med Chem 10 1741... 

Very recently, (3-lactam antibiotics have been shown to offer neuroprotection by increasing glutamate transporters expression via gene activation [15] in addition, the discoveries of new biologically active (3-lactams such as cholesterol acyl transferase inhibitors [16-18], thrombin inhibitors [19], human cytomegalovirus protease inhibitors [20], matrix-metallo protease inhibitors [21], inhibitors of human leukocyte elastase (HLE) [22, 23] and cysteine protease [24, 25], and apoptosis inductors [26, 27] have provided much needed motivation for continuous development of new (3-lactam systems. 

Both substituted 3-amino-2//-l-benzothiopyrans <1995EP648741> and 4-acyl-4-hydroxy-2//-l -bcn/othiopvrans <2001W02001087879> inhibit thrombin activity and thus behave as anticoagulants and 4-(SCH2CONR2) derivatives of 3,4-dihydro-2//-l-benzothiopyran 1-oxide have been claimed as treatments for excessive sleepiness and fatigue <2005USP2005228040>. 

The active site structure of trypsin-like enzymes is considered to be very similar to that of bovine trypsin, yet little is known about them. Refinement of these structures is important also for the purpose of designing physiologically active substances. With a view to comparing the spatial requirements of active sites of these enzymes, dissociation constants of the acyl enzyme-ligand complex, K-, which were defined before, were successfully analyzed By taking advantage of inverse substrates which have an unlimited choice of the acyl component, development of stable acyl enzymes could be possible. These transient inhibitors for trypsin-like enzymes could be candidates for drugs. In this respect, the determination of the deacylation rate constants for the plasmin- and thrombin-catalyzed hydrolyses of various esters were undertaken 77). 

Alam L Silver Ml. Metabolism of l-aUcyl-2-acyl-GPC in human platdets in response to stimulatim by thrombin. Hnoiid> Res 1987 45 311-322... 

Mahadevappa VG, and Holub BJ. (1983). Degradation of different mdecularspedes of idnsphatidyl-inoshol in thrombin stimulated human platelets Evidence for preferential deradation of 1-acyl 2-aradiidonoyl species. J. Biol. Chem. 258,5337-5339. 

FIGURE 9.98 Events taking place in the plasma membrane on stimulation of a cell. Phospha tidylinositol (Pi) and more hi ly phosphoiylated versions of this lipid account for 2 to 8% of the lipids of the plasma membrane of eukaryotic cdIs. The inositol 1,4,5 triphosphate (fP3) moiety of phosphatidyl inositol- 4,8-di phosphate may be hydiulyitec from this lipid immediately after the cell is stimulated. For example, the stimulation of platelets by thrombin or the islets of the pancreas by glucose is followed by the release of 1P3 into the cytoplasm. In some cells, arachidonic acid is hydrolyzed from l-acyl-2-arachidonyl-glyceroL which can Support a burst of prostaglandin. synthesis. 

The phospholipase As from Vinera Berus venom was purified and its properties were determined77 (mol. wt. 13,400 and isoelectric point 9.2). This enzyme hydrolyses the acyl group at the 2 position of phospholipid, thereby reducing the procoagulant activity of the phospholipoprotein cofactor in thrombin production. 

To demonstrate the effect of the active-site acylated enzyme, we prepared the benzoyl derivatives of bovine thrombin [22,23]. If benzoyl-thrombin is added to blood plasma, the clotting process is retarded in accordance with the deacylation rate (Fig. 7). 

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