BIOCHEMISTRY - L. Stryer - 1984

VOLUME 1

PART I. CONFORMATION AND DYNAMICS

CHAPTER 8. ZYMOGEN ACTIVATION: DIGESTIVE ENZYMES AND CLOTTING FACTORS

8.24. Prothrombin is activated by Factor X on the phospholipid surface of platelets.

The cascade nature of Blood clotting is shown in Fig. 8.35. Prothrombin is activated by a proteolytic enzyme called Factor X_a. For convenience, blood clotting factors are designated by Roman numerals. The letter "a" indicates that the factor is in its active form. Thus, prothrombin is converted to Thrombin by the proteolytic action of Factor X_a. This conversion (prothrombin → thrombin) is accelerated by Factor V. Factor V is not an enzyme; it acts as a modifier protein.

As a result of a vitamin K-dependent carboxylation reaction, glutamate (which weakly chelates Ca^{2 +}) is converted into γ-carboxyglutamate, a strong chelating agent. By binding to Ca^{2 +}, prothrombin is anchored to the phospholipid membranes of platelets that accumulate at the site of injury. The Functional Significance of prothrombin binding to phospholipid surfaces is that prothrombin is brought into close proximity with Factors X_a and V, which accelerates its activation by more than 10⁴-fold. At this stage of activation, the N-terminal fragment of prothrombin, which contains the Ca^{2 +}-binding sites, is cleaved off. As a result, thrombin is released from its bond with the phospholipid surfaces and, in its free form, can activate plasma fibrinogen.

Class="center">Fig. 8.35. The final stages of fibrin clot formation. These three reactions constitute the common pathway of Blood Coagulation. Both the intrinsic and extrinsic pathways contribute to the activation of Factor X.

How is Factor X activated? Its activation step marks the convergence of the intrinsic and extrinsic pathways of blood coagulation. Both pathways lead to the generation of Proteolytic Enzymes that activate Factor X.

8.25. Hemophilia and other bleeding disorders have helped elucidate several early steps in blood clot formation

The biochemical Study of the Cytology/cytology/16.html">Early stages of blood clotting proved to be much more difficult than that of the final stages, because the factors involved in the initial steps are present in the blood in very small amounts. For instance, while the concentration of fibrinogen is 3 mg per ml of blood, that of Factor X is only 0.01 mg. The concentrations of several other factors involved in the very first steps of clotting are even lower. Furthermore, these factors are extremely labile. For these reasons, an alternative approach—The Study of patients with bleeding disorders—proved to be highly fruitful.

The most famous disease of this type is hemophilia. This bleeding disorder is inherited as a sex-linked recessive trait. Heterozygous females transmit the defective Gene to their offspring, while remaining asymptomatic carriers themselves. Queen Victoria was a famous carrier of hemophilia, passing the defective gene to the royal families of Prussia, Spain, and Russia (Fig. 8.36).

Tsarevich Alexei was born in 1904. He was the sole heir to the Romanov dynasty, which had ruled Russia since the 17th century. By the time the Tsarevich was born, Tsar Nicholas II and Empress Alexandra, a granddaughter of Queen Victoria, already had four healthy daughters. Just six weeks after the Tsarevich's birth, his father noted in his diary: "This morning, for no apparent reason, little Alexei's navel began to bleed. The bleeding continued with short intervals until evening." The symptoms worsened when Alexei began to crawl and walk; large, swollen bruises began to appear on his arms and legs. The hematomas grew larger, but doctors were powerless to alleviate the boy's condition. In desperation, the Empress turned to Rasputin, who had a reputation as a healer. No one in history had less understanding of the Molecular Basis of hemophilia, and no one in history profited more from it. For many years, Rasputin wielded immense influence at the Russian court because the Empress blindly believed in his healing powers.

Fig. 8.36. Pedigree of hemophilia in the royal families of Europe. The diagram shows all of Queen Victoria's children and selected grandchildren and great-grandchildren. Females are represented by circles, males by squares; hemophiliacs are shown in red.

How could the marriage between Nicholas and Alexandra have taken place when it was already known that her brother, nephews, and uncle suffered from hemophilia? As early as 1873, the French physician Grandidier recommended "that all members of a bleeder's family should abstain from marriage." Moreover, the hereditary nature of this disease had been pointed out as early as 1803 by John Otto:

"About seventy or eighty years ago, a woman by the name of Smith, settled in the vicinity of Plymouth, New Hampshire, and transmitted the following idiosyncrasy to her descendants... It is a surprising circumstance that males only are subject to this strange affection, and that all of them are not liable to it... Although females are exempt, they are still capable of transmitting it to their male children."

As J. Haldane wrote, "royalty are carefully shielded from contact with unpleasant realities... The hemophilia of Tsarevich Alexei was a symptom of the royal house's complete failure to grasp reality."

8.26. The Intrinsic Pathway of Blood Coagulation

In hemophilia, the intrinsic pathway of blood coagulation is impaired (Fig. 8.37)—a protein called antihemophilic factor (Factor VIII) is either completely absent or its activity is severely reduced. This protein, acting together with Factor IX_a (a proteolytic enzyme), activates Factor X. The antihemophilic factor itself is not an enzyme, but a modifier protein.

Fig. 8.37. The intrinsic pathway of blood coagulation. Inactive forms of clotting factors are shown in red, activated forms in yellow. Activated factors catalyze the activation of the subsequent ones.

The story of the discovery of Factor IX is intriguing. A boy named Stephen Christmas suffered from a bleeding disorder that was clinically indistinguishable from classic hemophilia in both symptoms and inheritance pattern. As in hemophilia, the clotting time of his blood in a Glass test tube was prolonged. When his blood was mixed with that of a hemophilic patient, the clotting time of the mixture was surprisingly found to be nearly normal. This clearly demonstrated that, despite identical clinical presentations, the molecular defects in Stephen Christmas and the hemophilic patient were different. These two patients lacked different clotting factors; otherwise, their plasma would not have been capable of mutual complementation. Thus, a new factor was discovered, designated as Factor IX, or Christmas factor. In general, the complementation test is a reliable experimental method to determine whether the same or different components are missing in two compared inactive systems. The value of this method is that it allows individual components of a system to be numbered even before they can be isolated and purified. The complementation test is widely used in genetic studies of Viruses and Bacteria.

Christmas factor is separated by two steps from the initiation reaction of the intrinsic pathway. The trigger for this sequence of reactions is the contact of Factor XII with a foreign surface. Factor XII_a, activated by this contact, acts together with kallikrein and kinin to convert Factor XI into its active form, Factor XI_a, which in turn activates Christmas factor (IX). At each of these activation steps, zymogens are converted into active enzymes.