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Figure 9.6. Proteolytic cleavage of prothrombin by factor Xa, yielding active thrombin. While prothrombin is a single-chain glycoprotein, thrombin consists of two polypeptides linked by what was originally the prothrombin intra-chain disulphide bond. The smaller thrombin polypeptide fragment consists of 49 amino acid residues, while the large polypeptide chain contains 259 amino acids. The N-terminal fragment released from prothrombin contains 274 amino acid residues. Activation of prothrombin by Xa does not occur in free solution but at the site of vascular damage
to aggregation, therefore promoting soft clot formation (Figure 9.7). The soft clot is stabilized by the introduction of covalent crosslinkages between individual participating fibrin molecules. This reaction is catalysed by factor XIIIa, as shown in Figure 9.8.
Factor XIII is present in both plasma and platelets. Plasma factor XIII is a 320 kDa tetramer, composed of two (70 kDa) a-chains, and two (90 kDa) b-chains. The platelet form of factor XIII
Soft clot of fibrin
Figure 9.7. Diagrammatic representation of the fibrinogen molecule and its conversion to the soft clot of fibrin. Reproduced (in modified form) by permission from Textbook of Biochemistry with Clinical Correlations (3rd Ed.) Devlin (1992). This material is used by permission of John Wiley & Sons, Inc.
BLOOD PRODUCTS AND THERAPEUTIC ENZYMES 365
Figure 9.8. Formation of a hard clot via the action of factor XIIIa. This activated factor displays a transglutaminase activity, which catalyses direct formation of a covalent linkage between a glutamine side-chain of one fibrin molecule and a lysine side-chain of a second fibrin molecule. The resultant highly crosslinked hard clot is both tough and insoluble
is composed solely of the two a-chains (i.e. a 140 kDa a-dimer). Both forms of factor XIII are activated upon proteolytic cleavage by thrombin (factor IIa), which hydrolyses a single arg-gly bond in the NH2 terminal region of the a-chains. This generates a 73 amino acid peptide and a modified a-chain (a).
In the case of platelet-derived factor XIII, the resultant product (a)2, is the activated form. Thrombin action on plasma-derived factor XIII generates an a02b2 dimer, which is devoid of transglutaminase activity. However, in the presence of Ca2 + , the a'b chains dissociate, yielding the biologically active a 2.
Genetic defects characterized by (a) lack of expression, or (b) an altered amino acid sequence of any clotting factor can have serious clinical consequences. In order to promote effective clotting, both intrinsic and extrinsic coagulation pathways must be functional, and the inhibition of even one of these pathways will result in severely retarded coagulation ability. The result is usually occurrence of spontaneous bruising and prolonged haemorrhage, which can be fatal. With the exception of tissue factor and Ca2 + , defects in all other clotting factors have been characterized.
Table 9.6. Recombinant blood coagulation factors which have been approved for the management of coagulation disorders
Product (trade name) Company Indication
Bioclate (rhFactor VIII) Centeon Haemophilia A
Benefix (rhFactor IX) Genetics Institute Haemophilia B
Kogenate (rhFactor VIII) Bayer Haemophilia A
Helixate NexGen (rhFactor VIII) Bayer Haemophilia A
NovoSeven (rhFactor VIIa) Novo-Nordisk Some forms of haemophilia
Recombinate (rhFactor VIII) Baxter Healthcare/Genetics Institute Haemophilia A
ReFacto (B-domain deleted Genetics Institute Haemophilia A
Up to 90% of these, however, relate to a deficiency in factor VIII, while much of the remainder is due to a deficiency in factor IX.
Such clotting disorders are generally treated by ongoing administration of whole blood or, more usually, concentrates of the relevant coagulation factor purified from whole blood. This entails significant risk of accidental transmission of blood-borne disease, particularly hepatitis and AIDS. In turn, this has hastened the development of blood coagulation factors produced by genetic engineering, several of which are now approved for general medical use (Table 9.6).