RESEARCH
Blood Clotting Goes Through Four Steps Final and crucial step in mechanism is cross-linking of fibrin molecules by transamidation to yield insoluble, hard clots More details on the role of thrombin in the chemistry of blood clotting are becoming apparent as a result of continuing research by Dr. Laszlo Lorand and his co-workers at the biochemistry division of Northwestern University's chemistry department (Evanston, 111.). They now find that thrombin, the enzyme which catalyzes the hydrolysis of fibrinogen to fibrin, forms an acyl-enzyme intermediate during blood clotting. Fibrinogen is a bloodplasma protein produced by the liver; it is the key gel-forming substance in vertebrate blood clotting. Last year, Dr. Lorand and his group demonstrated that thrombin activates the enzyme which catalyzes the crosslinking of fibrin in the last stage of blood clotting. This finding and the recent work are only two of a series of results obtained by the Northwestern group in its studies of the extremely complex chemistry of blood clotting. A fuller understanding of the chemistry of blood clotting could aid in the eventual development of drugs to control clotting. Also, greater knowledge of blood clotting should advance protein chemistry in general, because fibrinogen and fibrin show many similarities with other fiber-forming structural proteins (such as epidermin, the skin protein ). In the early stages of blood clotting, an activator, thromboplastin, converts prothrombin (a precursor present in blood plasma) to thrombin. But Dr. Lorand is concerned only with the later stages of clotting—those which occur after the formation of thrombin. In his work during the past 15 years, he has separated this terminal clotting into four steps. Each of these steps is now being studied in detail at Northwestern. In the first step, fibrinogen hydrolyzes under the influence of thrombin into fibrin and fibrinopeptide fragments. In the second, fibrin forms soft, fibrous networks (soft clots) which can be readily dispersed. Next, thrombin activates the fibrin-stabiliz38
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stem largely from their similar structures, Dr. Lorand says. He points out that it is already known that chymotrypsin and trypsin are much the same structurally. The full structure of thrombin is not yet known, however. Thrombin hydrolyzes substrates by the same mechanism that has already been shown by other workers for chymotrypsin and trypsin. The process involves an acyl-enzyme intermediate. After a rapid adsorption equilibrium with the enzyme ( E H ) , the substrate ester (RCOOR') acylates the enzyme to form the acyl-enzyme intermediate ( R C O E ) . At the same time, alcohol (R'OH) is eliminated. The intermediate then reacts with water and deacylates into acid (RCOOH) and free enzyme. Thus it is a three-step mechDr. Laszlo Lorand Study may lead to new type of drugs
fcCOofc ing factor ( F S F ) , an enzyme precursor present in blood plasma. Finally, fibrin in the networks cross-links under the influence of the activated FSF to give the final, hard clots. This crosslinking is achieved by a transamidating mechanism. Hydrolysis. The hydrolytic reaction was discovered by Dr. Lorand in 1951. Recognition of thrombin as a proteolytic enzyme in this reaction is the key to the present understanding of the chemistry of blood clotting. Subsequent studies of how thrombin catalyzes the hydrolysis of synthetic esters and other synthetic substrates have shown that thrombin behaves much like chymotrypsin and trypsin. These proteolytic enzymes are found in digestive systems. Dr. Lorand explains that the hydrolysis of relatively simple synthetic esters is much easier to follow than that of proteins. And, he adds, it makes good sense to assume that hydrolyses of both synthetic esters and proteins follow identical paths. The similar reactivities of chymotrypsin, trypsin, and thrombin must
+
EH
IÎ
1. Adsorption
RCOOR' γ
Rcoe + tHoH \ * RCOOH
^ +
6H
2. Acylation
ROM 3. Deacylation
EH
If deacylation becomes the ratedetermining step, there should be an initial burst of alcohol production be fore steady-state recycling of the en zyme can take place. The extent of this burst will depend on the concen tration of the enzyme. The size of the burst should thus measure the en zyme's functional normality. The Northwestern scientists have detected a burst of nitrophenol pro duction during the hydrolysis of nitrophenylesters under the influence of pure human thrombin. This burst confirms the acyl-enzyme mechanism,
they believe. From its extent, the sci entists estimate the maximum weight of the enzyme's functional unit to be about 32,000. Dr. Lorand notes that, although thrombin's action is, in many ways, similar to that of other proteolytic en zymes, it also shows an apparently unique specificity when reacting with large protein molecules. When it first reacts with the huge fibrinogen mole cule, for instance, it cleaves only four particular peptide bonds. This spec ificity, which is critically important physiologically, is not yet fully under stood. Aggregation. The fibrinopeptide fragments, split from fibrinogen on hy drolysis with thrombin, contain a pre ponderance of acidic amino acids. Thus they are negatively charged at neutral pH. Removal of these frag ments from the solution triggers the aggregation of the remaining fibrin protein into a loose fibrous network (soft clots). Hence it might be the electrostatic repulsion between these fragments that prevents fibrinogen from aggregating in normal blood, Dr. Lorand points out. He explains that loss of the fibrino peptide fragments (about 3 % by weight of the original fibrinogen) does not significantly change the main structural feature of the parent pro tein. There is no major unfolding or rearrangement of the molecule when fibrinogen is hydrolyzed to fibrin. Activation. The clotting of blood is by no means complete with the ap pearance of these loose fibrous net works. Such soft clots are too weak to permanently prevent bleeding. To form hard, strong clots, the fibrin in these loose networks must be crosslinked by FSF. But first, the FSF must be activated by thrombin. The Northwestern group deter mines the activity of FSF by methods based on the solubility of blood clots. They measure the difference in solu bility between the stabilized (exposed to activated FSF) and the nonstabilized clots. They use a 1% solution of monochloroacetic acid as solvent. The amount of fibrin that doesn't dissolve after 18 hours exposure to the acid is taken as an index of stabilization or cross-linking. The greater the insolu ble residue, the greater the cross-link ing in the clot, and the more active the FSF has been. In these tests, FSF is incubated with thrombin for a known time and the reaction is then quenched with a
Thrombin Has Two Roles in Final Stages of Blood Clotting I. Fibrinogen (blood plasma protein)
-
Hydrolysis
_ • Thrombin (proteolytic enzyme)
Fibrin + Fibrinopeptides
14 II.
Aggregation
τI Fibrin Aggregates (soft, soluble clots)
III. Activation
FSF
•(FSF)*
(Fibrin-Stabilizing Factor)
IV. Cross-Linking
Thrombin, Ca++
Τ Cross-Linked Polymer (final, hard clots)
Final, Cross-Linking Step Is Transamidation
γ
Il
-^ +
'->,
Os?)* ++ H Ca
+ HX C-M
^$-*'
I
In transamidation, terminal amino groups of one fibrin molecule react with certain carbonyls of another. Activated FSF and Ca+2 are needed for the condensation
material which competes with FSF for thrombin. A solution of purified fibrin is subsequently added, and the clots are allowed to cross-link. At predetermined times, the solubility of the clots is measured. The experiments show that FSF is only the precursor of the active prin ciple (activated F S F ) , and that thrombin is needed to activate it. The tests also prove that calcium ions are essential both to the activation of FSF and to the cross-linking of fibrin. With ( F S F ) * and fibrin, and without calcium ions and thrombin, only com pletely soluble clots are formed. Dr. Lorand says that knowledge of how to activate FSF by thrombin and calcium ions will permit studying the action of the cross-linking enzyme on substrates other than fibrin. Cross-Linking. In the final crosslinking of fibrin by activated FSF, the terminal amino groups of one fibrin molecule react with certain carbonyl functions of another. Dr. Lorand ex plains that such a mechanism suggests the possible existence of two types of inhibitors.
One type of inhibitor would include compounds that imitate the amino groups of the donor fibrin molecule. The other type would simulate the acceptor carbonyl function of the at tacked fibrin. Such inhibitors would prevent cross-linking between fibrin molecules by masking their active sites. The Northwestern chemists have found many such inhibitors. The donor kind is typified by glycine ethyl ester, the acceptor type by carbobenzoxyl-L-asparagine amide. These inhibitors provide a means of labeling the active cross-linking sites of fibrin. Thus, these sites can be identified. The Northwestern group is now working to find out which chains of the fibrin molecule contain these sites. From a biological point of view, inhibition of fibrin cross-linking pro vides a method for influencing the final structure of blood clots with out interfering with their initial for mation. This concept may promise development of a new class of blood clotting inhibitors as drugs, Dr. Lorand hints. AUG.
9,
1965
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