Chemistry and Biology of Proteins - F. Haurowitz 1953

Albumins, globulins and other soluble proteins
Fibrinogen and fibrin

Blood Coagulation is caused by The conversion of the soluble plasma protein fibrinogen into the insoluble protein fibrin. A vast number of studies have been devoted to investigating The Mechanism of this process, which is one of the most remarkable biological phenomena. Despite this, the question remains not fully resolved to this day. The conversion of fibrinogen into fibrin is triggered by the action of a specific enzyme, Thrombin. Blood Plasma contains a precursor of thrombin, prothrombin, which is converted into active thrombin under METABOLISM/18.html">The Influence of a thromboplastic substance (thrombokinase). Thus, the blood clotting process consists of two phases:

Class="center">

Prothrombin is a globulin. Upon Fractionation of blood serum with ethyl alcohol, it is found in fraction III-2. It can be purified by adsorption on colloidal calcium phosphate [51], aluminum hydroxide [52], or magnesium hydroxide [53]. To extract prothrombin from the adsorbents, a phosphate buffer (pH 8) is used, or magnesium hydroxide is treated with carbon dioxide. The purest prothrombin preparations contain 2,000–2,500 units per 1 mg of protein [53].

Prothrombin is converted into the active enzyme thrombin under the influence of thromboplastin—a substance present in Blood Platelets, Lungs, Brain, and other Organs. Rabbit brain is commonly used as a source of thromboplastin in clinical blood tests for prothrombin content. Thromboplastin is apparently contained in cellular granules, as high-speed centrifugation detects it in the precipitate [54]. Thromboplastin is a loosely bound complex of protein with ribonucleic acid and acetalphosphatide [55]. This complex can be split into a Water-soluble protein and an insoluble lipid. Although the latter is in many respects similar to cephalin, substituting it with synthetic cephalin does not allow the conversion of prothrombin into thrombin to proceed [55].

The MECHANISM OF ACTION of thromboplastin is not yet entirely clear. Most authors believe that thromboplastin is an enzyme. This is supported by the fact that Trypsin is also capable of causing the conversion of prothrombin into thrombin [56]. From this perspective, the old name of this substance—thrombokinase—is fully justified [55]. On the other hand, it should be noted that thromboplastin and calcium react with prothrombin in stoichiometric ratios and, therefore, do not behave as catalysts in this case [57]. It has long been known that the presence of Calcium Ions is necessary for blood clotting. The blood clotting process can be prevented by adding oxalates, citrates, or sodium fluoride to the blood, i.e., substances capable of binding calcium ions. Calcium can also be removed from plasma by adsorption on Amberlite IR100; if calcium salts are added to such plasma, it regains The ability to form a clot [58].

Most researchers believe that calcium is required for The first phase of the clotting process, i.e., for The formation of active thrombin from prothrombin [59, 60]. The conversion of fibrinogen into fibrin occurs under the influence of thrombin even in the absence of calcium. The Role of calcium ions or calcium complexes [61] in the first phase of clotting is not yet completely clear. It can be hypothesized that calcium either activates thromboplastin or inhibits the action of a specific antiprothrombin [62]. Calcium is not required for snake venom clotting; this is confirmed by the fact that snake venom clotting can also occur in the presence of oxalic acid [63].

Some authors view the conversion of prothrombin into thrombin as an autocatalytic process, specifically as a chain reaction. This reaction begins with the formation of thrombin from prothrombin under the influence of thromboplastin and calcium, and then continues under the catalytic action of the newly formed thrombin itself [60], much like The process of pepsinogen conversion into Pepsin (see Chapter XII). The PHYSICOCHEMICAL PROPERTIES OF thrombin are similar to those of prothrombin. Thrombin is extracted from the fibrin clot with salt solutions and precipitated from blood serum with alcohol. Purest thrombin preparations are obtained by the action of thromboplastin on prothrombin in the presence of calcium salts [64]. The Molecular Weight of thrombin is approximately 77,000, and that of prothrombin is 140,000; it has not yet been established whether one molecule of prothrombin yields one or two molecules of thrombin upon its conversion into the active enzyme [50]. Dry preparations of pure thrombin are very stable and retain their activity at room Temperature for many months [50].

When thromboplastin acts on a pure prothrombin preparation, thrombin formation proceeds very slowly. However, the process can be significantly accelerated by adding another component, the so-called Ac-globulin (from the word accelerator). This protein was recently discovered in blood plasma; using various adsorption Methods, it was successfully obtained in a purified state [65].

From the data presented, it can be concluded that the first phase of clotting—the formation of thrombin from prothrombin—is a very complex process, the details of which are still insufficiently elucidated.

The second phase of blood coagulation—the formation of fibrin from fibrinogen—is also catalyzed by thrombin. Neither fibrinogen nor fibrin possesses enzymatic activity. In its properties, fibrinogen belongs to the globulin group. The fibrinogen content in blood plasma ranges from 0.2 to 0.4%. Fibrinogen is precipitated from plasma by Treatment with low concentrations of ethyl alcohol or by adding an equal volume of a saturated sodium chloride solution to the plasma [66, 67]. Fibrinogen molecules, whose solutions have high viscosity, exhibit Flow Birefringence and have the shape of elongated ellipsoids [68]. The length of the ellipsoid is approximately 700 Å, and the axial ratio is 18:1. Highly pure fibrinogen preparations can be obtained from plasma by freezing and subsequent thawing [69].

The enzymatic Nature of the conversion of fibrinogen into fibrin is currently beyond doubt; however, the exact nature of the changes that the fibrinogen molecule undergoes remains unclear. There is reason to believe that the transition of fibrinogen into fibrin, similar to the Denaturation of native protein, consists of the unfolding of the peptide chains of fibrinogen molecules. During this unfolding, positive and negative groups of the fibrinogen molecules are exposed, which interact with each other to form a network of fibrin molecules; the individual chains of this network are linked by salt bridges [70]. This view is supported by the inhibition of fibrin clot formation by all substances capable of binding to positively or negatively charged groups of the protein molecule; such substances include heparin and formaldehyde, which react with amino groups [70], as well as basic Dyes capable of attaching to the acidic groups of fibrinogen [71]. It is quite possible, however, that heparin affects the first phase of the clotting process by acting as an antiprothrombin. If the conversion of fibrinogen into fibrin is indeed a denaturation process, then thrombin should be classified as a denaturase [72], i.e., an enzyme that catalyzes the Cleavage of weak bonds between individual peptide chains. This hypothesis is also supported by the fact that the action of thrombin on fibrinogen releases sulfhydryl groups in the latter [73]. It must be noted, however, that substances such as chloromercuric derivatives of benzoic acid, which selectively react with sulfhydryl groups, do not prevent fibrinogen clotting [50]. The hypothesis that fibrinogen clotting is associated with its denaturation is also supported to some extent by the inhibition of this process under high pressure (on the order of 800 atm) [74]. As is known, high pressure prevents the formation of Salt bridges between ionized groups, because this process, accompanied by the release of water from hydrated ionized groups, must also be accompanied by an increase in volume.

If thrombin acts on fibrinogen at pH 5.1, no visible changes in fibrinogen occur. However, fibrinogen is thereby converted into the so-called profibrin, which easily forms a fibrin clot at a neutral reaction [75]. These data also correspond to the assumption that the peptide chain of fibrinogen first unfolds and then undergoes polymerization to form a network of fibrin fibers.

It is well known that blood plasma does not clot for a long time when stored in paraffin-coated vessels; however, if stored in non-paraffinated Glass vessels, it clots very rapidly. This phenomenon indicates that contact with a glass surface accelerates clotting. The glass surface plays a similar catalytic role in certain chain reactions. It is natural to assume that clot formation also proceeds via a chain reaction type. The conversion of fibrinogen into an insoluble fibrin clot can also be accelerated by adding small amounts of ninhydrin [76].

Electron Cell/15.html">Microscopy of the fibrin clot has shown that fibrin fibers have periodic transverse striations, with distances between individual bands equal to approximately 250 Å [77]. The beaded shape of fibrin fibers suggests that they are formed by the longitudinal association of globular particles and that the polymerization process leading to the conversion of fibrinogen into fibrin consists of end-to-end joining of fibrinogen molecules. Otherwise, it is difficult to imagine the formation of a large fibrin clot from small amounts of fibrinogen [78]. Presumably, the structural unit of the fibrin clot is a cell composed of 10–14 fibrinogen molecules connected to each other by their ends along the longitudinal axis. In the initial loose fibrin clot, there is probably only a small number of lateral cross-links.

The formation of a fibrin clot is sometimes compared to the formation of a gel from a protein solution. However, the consistency and Structure of a fibrin clot differ significantly from those of typical gels. For example, a fibrin gel containing only 0.2–0.4% fibrin is a much looser formation than a gelatin gel; when a fibrin clot is whipped with a glass rod, the fibrin threads wind around the rod, causing the clot to sharply decrease in volume. A fibrin clot that initially occupied a volume of 100 ml can be reduced in this way to a volume of 1 ml. This phenomenon is used for the quantitative isolation and determination of fibrinogen and fibrin in blood plasma. The ease with which the fibrin network can be compressed to a small volume shows that fibrin fibers join together by their lateral surfaces as soon as they come into contact. Such mutual attraction of fibrin fibers leads to the formation of crystal-like aggregates of parallel fibers (Fig. 35) [78, 79]. The Nature of the Bonds Responsible for the end-to-end joining of fibrinogen molecules, as well as the nature of the lateral cross-links between fibrin fibers, has not yet been elucidated. However, it is certain that ionic bonds play an important role in clot formation. This is indicated by the ability of fibrin clots to adhere to the silicon crystal lattice on the glass surface, as well as the inhibition of plasma clotting when stored in paraffin-coated vessels. It is possible that ionic groups are released under the action of thrombin. True, we still know nothing definite about the nature of these ionic groups. We cannot even say whether they arise from the cleavage of salt bridges, peptide bonds, or some other reaction.

Fig. 35. Fibrin clot [80].