Prothrombin structure, activation, and biosynthesis.

Physiological Published

and Copyright

The American

Physiological

Reviews

by

Vol. 57, No. 1, January

Society

1977

Prothrombin Structure, Activation, and Biosynthesis JOHN

W. SUTTIE

AND

CRAIG

M. JACKSON

Department of Biochemistry, College of Agricultural and Life Sciences, University Wisconsin, Madison, Wisconsin; and Department of Biological Chemistry, Division of Biology and’Biomedica1 Sciences, Washington University School of Medicine, St. Louis, Missouri I. Introduction ....................................................... II. Prothrombin Structure and Activation. .............................. A. Development of current picture of prothrombin activation ......... B. Structural chemistry of prothrombin ............................. C. Other prothrombin activation system components ................. D. Prothrombin conversion to thrombin ............................. E. Possible mechanisms for prothrombin activation .................. F. Prothrombin activation by activators not derived from blood ....... G. Contribution of accessory components to Factor Xa in prothrombin activation.. .................................................... H. Speculations on a mechanism for organizing and localizing the prothrombin activation process and other stages in the coagulation cascade ........................................................... III. Biosynthesis of Prothrombin ........................................ A Indirect evidence for a prothrombin precursor protein ............. B Immunochemical evidence for a prothrombin precursor ............ c Isolation and characterization of abnormal prothrombin ........... D Characterization of y-carboxyglutamic acid ....................... E Isolation of rat liver prothrombin precursor protein ............... F In vitro prothrombin production - carboxylation ................... G. Molecular function of vitamin K .................................. H. Normal regulation of prothrombin concentration .................. I. Alternate theories of vitamin K action ........................... IV. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

of

1 3 3 8 24

28 31

33 34 41 43 44 46 47 48 49 50 52 54 54 55

INTRODUCTION

The formation of a blood clot follows a series of complex biochemical reactions that, except for the polymerization of monomeric fibrin, primarily involve transformations of precursors of proteolytic emymes from inactive to active forms. In contrast, however, to the well-understood, simple proteinase-

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catalyzed transformations of pancreatic proteinase zy Fmogens into their active forms, the zymogen activations of blood coagulation are catalyzed by multicomponent enzyme systems. The initial cascade models for blood clotting proposed by Macfarlane (263) and Davie and Ratnoff (85) first recognized the many similarities between zymogen activations occurring in the blood clottin .g process a.nd in the activ vation of the zymogens of pancreatic pro teinases and formalized them into a scheme for blood coagulation. Subsequent schemes have been revised to include the participation of the accessory components of the complex zymogen activators that act as “cofactors” to the activating proteinases and dramatically affect their catalytic activity (83, 172, 264). A modified version of the coagulation cascade that presents each of the zymogen activations as occurring in a discrete stage involving an enzyme complex is shown in Figure 1 (198). This conceptual model is adapted from the earlier proposals (85, 172, 263, 264) but differs by directing attention to the role of the accessory components in the multicomponent complexes. Such redirection of attention emphasizes the differences between the coagulation system and the classical zymogen activation systems and seems appropriate because, at our current level of understanding, it is the differences rather than the well-established similarities that are much more striking. In particular the large increases in activation rate that occur when . accessory compone nts are added to the coagulation pro teinases and the specific interactions of the vitamin K-dependent clotting factors with phospholipid membrane surfaces most dramatitally distinguish the coagulation proteinase zymogen activation systems from their pancreatic protease counterparts. Some recently demonstrated reactions, namely activation of Factor VII by Factor Xa and activation of Factor IX by Factor Xa, which interrelate products of the intrinsic and extrinsic Factor X activation systems (222, 389), and some of the reactions involved in the initial stage of Factor XII activation (396) have been omitted from this diagram. The penultimate stage of the coagulation process is the conversion of prothrombin to the proteinase thrombin. In addition to the proteinase, Factor Xa, which arises from the previous stage, prothrombin activation, involves three accessory components: phospholipid, Ca2+, and Factor V, the last in an “activated” form. During the course of investigation of the details of prothrombin activation and the role of the accessory factors in this process, it has become clear that the functions of Ca2+ and phospholipid in this system are related to specific properties of prothrombin and Factor Xa conferred on them by a vitamin K-dependent step during their synthesis (116). These two proteins, as well as Factors VII and IX, require vitamin K for their formation, and these four proteins have long been called the “vitamin K-dependent” clotting factors. Only during the last few years has sufficient evidence accumulated to demonstrate conclusively how vitamin K functions and how vitamin K function is related to the calcium-dependent association of these proteins to phospholipids. It is the recent elucidation of the chemistry and biochemistry of prothrombin, the generation of thrombin from prothrombin


January

1977

3

PROTHROMBIN

including the participation of the accessory factors role of vitamin K in the biosynthesis of prothrombin addressed. II.

PROTHROMBIN

A. Development

STRUCTURE

of Current

AND

Picture

in this process, and the to which this review is

ACTIVATION

of Prothrombin

Activation

Although prothrombin was only a hypothetical substance in it, the “classical theory” of blood coagulation proposed by Morawitz (321) was basically a theory of prothrombin activation: prothrombin

tissue

thrombokinase extract, thromboplastin

’ thrombin II I

fibrinogen-

fibrin

The large volume of work published on prothrombin activation since the proposal of this classical theory precludes a comprehensive treatment. However, we do attempt to trace the development of the current concept by citing landmark experiments as seen in retrospect in order to acknowledge both the original work and original ideas that form the foundation for the current picture. 1 The unambiguous demonstrations that thrombin action on fibrinogen (44, 99, 236, 253, 255) and prothrombin activation by trypsin (100) were proteolytic processes undoubtedly led the way to the contemporary view not only of prothrombin activation but of the entire coagulation process as a series of limited proteolytic cleavages of enzyme precursors (85, 263). Possibly more important, however, is the fact that these observations provided a simple view of how the coagulation process and specifically prothrombin activation might occur and thus provided an explicit basis for biochemical investigation of the clotting process. Prior to these demonstrations, a continuing debate existed about the nature of the prothrombin activator and 1 Even with the advantage that implicitly makes judgments of hindsight, an approach about what consti tutes a landmark experiment or observation is predisposed to omitting some of the work that had considerable impact at the time of its presentation. In addition, when viewed in retrospect the landmark observations of the past may not have been as influential in their time as it now appears that they should have been. As pointed out by Stent (471) in an essay entitled “Prematurity and Uniqueness in Scientific Discovery” recognition of the significance of many first observations, especially those that introduce completely new ideas into biology, frequently must await the development of a “context” within which these observations and ideas can be generally understood. This seems no less true in blood coagulation biochemistry, which had to await the development of other areas of protein and enzyme biochemistry, particularly pancreatic proteinase zymogen activation biochemistry, than in the contemporary molecular biology and molecular genetics discussed by Stent. The problem of “impact of discovery” specifically as seen in the development of the prothrombin concept in the classical theory of blood coagulation has been discussed in an excellent short review by Beck (35).


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Vohme

0’ m

FIG. 1. Staged cascade model for blood coagulation system. At each stage, except for the 2 initial stages of intrinsic and extrinsic systems, a proteinase, in conjunction with accessory components of the activation complex, converts the substrate (enclosed by rhombus) from an inactive to a highly active proteolytic enzyme, which then becomes the proteinase in the subsequent stage. Four components of the 6 coagulation factors enclosed in rhombi are “vitamin K-dependent” proteins: Factor VII, Factor IX, Factor X, and prothrombin. Recent discussions of the 2 initial stages and the nature of activation processes associated with them can be found in the monograph edited by Reich et al. (396). Participation of accessory components in the early stages is less well defined than in the late stages. Putative accessory component labeled @ could be the surface activator of Factor XII. A plasma activity has been


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PROTHROMBIN

5

the kinds of processes that could give rise to thrombin but did not provide an explicit focus for experimental attack. Although proenzyme transformations (125, 304) were among the various alternatives that were discussed, stoichiometric complexation reactions (304, 350) and release of the prothrombin activator from an enzyme-inhibitor complex by the removal of the inhibitor (191) were more widely entertained to explain the coagulation process than proteolysis. Interestingly, recent observations on prothrombin and plasminogen activation by two activators of bacterial origin suggest that they function as activators simply via stoichiometric complex formation (see below) and th .us some of the earl .y ideas about possi .ble nonproteolytic activation processes are reappearing as explanations for contemporary observations. [For detailed discussions of these issues the reviews of Howell (191) and Milstone (312) should be consulted.] A proliferation of different names for the same substances and single names for preparations of highly variable composition (312, 523), in the past as well as the present, undoubtedly complicated and impeded understanding the coagulation and prothrombin activation processes. The development by Quick (385) of the quantitative “prothrombin” assay procedure must be considered a landmark, as it not only permitted quantitative assessment of known clotting reactions but also facilitated recognition of the fact that the classical theory could not account for many observation .s about clotting . Owren’s (361, 362) observations on a bleeding disorder in a young woman provided the first evidence for the existence ofa activation component (Factor V) required for prothrombin that was not even implicitly included in the classical theory. At about the same time, Fantl and Nance (122) reported that a component in plasma from which “prothrombin” (equivalent to all vitamin K-dependent coagulation factors) had been removed could accelerate prothrombin activation. This component, identified only by its separability from “prothrombin,” was undoubtedly the same component as that missing in Owren’s (362) patient. The labile, human plasma clotting factor of Quick (386) and the bovine plasma protein with ability similar to that of Owren’s Factor V to accelerate prothrombin activation described by Ware et al. (335, 511-514) are now known as Factor V. Owren’s factor in an “activated” form, called Factor VI, was later proposed to be the primary activator of prothrombin (363). This proposal, which was reintroduced in the original cascade models of coagulation (85, 263), subsequently was found to be incorrect. [Factor VI consequently is an unused factor number in the list of clotting factors designated by roman numerals (523).] Dur ing the same period that the discovery of these factors was expanding the number of components participating in prothrombin activation, described but not chemically characterized (409) that participates in Factor XI activation g). To date no accessory component has been identified that participates in Factor IX activation @. Evidence has been presented (410,506,507) that indicates that platelet involvement in the stages involving Factors XII, XI, and IX may be of primary importance in this segment of the cascade. A review of the entire coagulation process has been done by Davie and Fujikawa (84).


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work by Seegers and co-workers (414,422,427,437,442) on the isolation and purification of bovine prothrombin and studies on its conversion to thrombin (98,306,311,414) removed the last traces of doubt about the existence of prothrombin as a chemical substance instead of only a hypothetical precursor of thrombin as it was in the classical scheme. The idea that substances such as Ca2+ ions, lipids, and proteins could act in concert with a proteinase activator of prothrombin may be attributed originally to Nolf (312, 351). In his discussion of thromboplastic substances in 1908 (351), Nolf states of the materials from various tissue extracts [translated by Milstone (312)]: “they are only adjuvants . . . I propose to bring them altogether under the designation of thromboplastic substances.” That such a view was not altogether widely accepted, however, can be most easily seen from the many theories of coagulation in which lipid-containing tissue extracts were viewed as being involved with prothrombin in a stoichiometric complex that had thrombin activity rather than as adjuvants to an enzyme that alone could catalyze prothrombin activation, albeit more slowly [see the review of Howell (191)]. Mellanby and Pratt (305), Eagle (98), and Milstone (311, 312, 318) demonstrated that Ca2+ participated as an accessory component to enzymatic processes in both fibrinogen clotting and in the conversion of prothrombin to thrombin, rather than stoichiometritally, as had been viewed by some investigators. The basic relationship between lipid substances, Ca2+ ions, and prothrombin activation (which is now known to be directly related to vitamin K function; see below) was described by Eagle (98), Ferguson and Erickson (125), and Milstone (311). Milstone in 1951 (311) showed that phospholipid affected the rate of thrombin formation from prothrombin but not the amount of thrombin ultimately formed and that acceleration of prothrombin activation by phospholipid did not occur in the absence of Ca2+. Although the identification of three accessory factors or adjuvants to prothrombin activation (i.e., phospholipid, Ca2+, and Factor V) was made by approximately 1950, the identification of the enzyme postulated in the classical theory to activate prothrombin, which was implied to be a proteinase by the experiments of Eagle and Harris (loo), came much later. The earliest modern efforts directed toward identifying, isolating, and characterizing an enzyme that could catalyze thrombin formation from prothrombin were those of Milstone (311). Milstone demonstrated the presence of an activity in a plasma globulin fraction that could catalyze the conversion of prothrombin to thrombin in the absence of Ca2+ (311,313) and identified this component as Morawitz’s “thrombokinase” that had been postulated along with prothrombin in the classical theory. The identification of “thrombokinase” as a proteolytic enzyme by analogy with the earlier experiments of Eagle on trypsin activation of prothrombin (98, 100) was supported by the observation that the action of thrombokinase on prothrombin was inhibited by soybean trypsin inhibitor (314) and that thrombokinase could hydrolyze the synthetic trypsin substrate, p-toluenesulfonyl+ arginine methyl ester (315-317). During and after this period three other, completely independent lines of evidence developed supporting the exist-


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PROTHROMBIN

7

ence of a coagulation factor directly involved in prothrombin activation. Product I of Bergsagel and Hougie (42, 456-458), Factor X in an activated form (121, 186,265,370,473), and autoprothrombin C (234,295,296) were all found to possess the capacity to convert prothrombin to thrombin. By the late 1960’s both the functional and structural similarity of these variously named prothrombin-activating enzymes and their zymogens had come to be generally recognized (93, 121, 131, 132, 196, 201, 202, 249, 302, 318, 370, 387, 388, 424, 430, 431, 434, 440), although some ambiguity existed as to the certainty of the identity of the differently named variants of activated Factor X as a result of proteolytic degradation of the primary form of the enzyme (93, 388). Although now of only historical interest, considerable controversy existed during this same period about the origin of Factor X and some other barium salt-absorbable (now known to be vitamin K-dependent) clotting factors. Seegers and co-workers (296, 419, 424) proposed that Factor X and Factor Xa (called by them autoprothrombin III and autoprothrombin C, respectively) were derived from the same protein molecule as prothrombin. However, substantial evidence based on physical, chemical, and immunological (12, 248, 474) properties of Factor X argued against this proposal. The recent publication of the complete amino acid sequences for both prothrombin (182, 275, 276, 397) and Factor X (103,496) has now completely eliminated this hypothesis. The development of the current view of how the accessory factors, Factor V, phospholipid, and Ca2+, participate in prothrombin activation has been perhaps the most confused aspect of the research effort devoted to this problem. In retrospect, the views expressed by Milstone (314, 318), who showed that, in the absence of “thrombokinase,” Ca2+ (3ll), phospholipid (311, 313), and the accelerator protein (Factor V) (314) could not alone or in combination convert prothrombin into thrombin, come extremely close to those of the current picture, although they do not seem to have been widely accepted at the time. The proposals presented in the cascade or waterfall (85, 263) models for blood clotting undoubtedly did . more than even the classical theory . to promote investigation of the clotting mechanism bY providing testable hY potheses about the zymogen activation steps in the reaction sequence. Although the original cascade (263) or waterfall (85) models for blood coagulation presented the clotting process as a linear sequence of reactions (85, 263, 264) in which Factor Xa (“thrombokinase”) was proposed to activa te Factor V, which then subsequently activated prothrom .bin, revision soon occurred that led to a return to the view derived from Milstone’s work that Factor Xa was the specific proteolytic activator of prothrombin. Experiments showing that no prothrombin activation occurred if the proteolytic activity of Factor Xa was inhibited by diisopropylfluorophosphate, even after preincubation of the Factor V with active Factor Xa to “activate” the Factor V (29), made it virtually certain that Factor Va could not be the primary activator of prothrombin. This conclusion was supported by the results of detailed kinetic investigations on prothrombin activation (170) and together with the inhibition experiments


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paved the way to the return of the view (311,313,314,473,474) that Factor V was acting as an accessory factor in conjunction with phospholipid and Ca*+ to promote Factor Xa-catalyzed activation of prothrombin. The discovery that some of the results of the experiments on which the linear-sequence hypothesis was based (53-55) reflected not only the action of a plasma coagulation component but also the action of an enzyme of Russell’s viper venom that was used as a tool in the experiments and activates Factor V (see below) (378) clarified the confusion generated about the primary prothrombin activator in the original cascade model. At about the same time as experiments forcing revision of the linear cascade were being reported, studies of how the accessory components such as Factor V, phospholipid, and Ca*+ ions might interact and thus participate in thrombin formation were being carried out independently of the question of their functional roles in prothrombin activation. These studies demonstrated that pairwise associations exist between phospholipid (in the form of lipid aggregates that provide a membranelike surface onto which proteins can adsorb) and Factor Xa in the presence of Ca*+ ions (68, 69, 118, 367, 370); between Factor V (Va?) and phospholipid (68, 69, 118, 171, 220); and between prothrombin and phospholipid, also in the presence of Ca*+ ions (28, 59, 118, 145). These observations suggested molecular mechanisms by which all the reactants of the prothrombin-activating system could be collected onto a common phospholipid surface (141, 145, 168, 171, 220) and provided a mechanism by which phospholipid could participate in clotting. The details of these interactions and how they are related to the structures of the components have become known only very recently. B. Structural

1. Purification

Chemistry

of Prothrombin

and general

characterization

Since most of our detailed knowledge of prothrombin chemistry and biochemistry has been derived from the study of bovine prothrombin, this review focuses primarily on bovine prothrombin and presents comparisons between bovine prothrombin and other prothrombins when data are available. Although prothrombin is a relatively minor component among the blood plasma proteins [less than 0.2% (10,272,470)], it can be isolated readily along with the other “vitamin K-dependent” clotting factors, Factors VII, IX, and X, by adsorption onto a variety of insoluble barium and other metal ion salts (149, 320, 394, 420, 493, 494, 504). Subsequent separation of homogeneous prothrombin from the other insoluble-salt-adsorbable (vitamin K-dependent) clotting factors is more dif&ult and requires high-resolution chromatographic (17, 75, 120, 194, 268, 269, 308, 311, 319, 359, 426) and/or electrophoretie techniques (371). Bovine prothrombin is relatively easily isolated in homogeneous form; human prothrombin and the other insoluble-salt-adsorbable proteins from bovine and particularly human plasma are only isolable in


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PROTHROMBIN

9

a comparable state of homogeneity to prothrombin by high-resolution electrophoresis or “affinity” chromatography techniques (91, 92, 229, 244, 449, 485, 486, 487). Because the sensitive, high-resolution protein electrophoretic techniques that permit de tection of these other components in crude prothrombin (origin .ally equated to barium salt-ad sorbable protein) preparations have only been available during the last decade, much of the early work on both bovine and human prothrombin chemistry was actually carried out on inhomogeneous protein preparations. As a result, many interpretations of early experiments (416, 417, 438) on prothrombin activation have had to be modified or abandoned because of effects due to the presence of the unrecognized contaminating clotting factors. Bovine prothrombin in its usually isolated form is a glycoprotein (254, 270, 309, 413) with a molecular weight of 72,600 t ca. 700, with carbohydrate accounting for 11% of the glycoprotein mass (Tables 1 and 2). During activation bovine prothrombin is converted to thrombin plus protein fragments that are soluble in trichloroacetic acid (TCA) (254) and have relatively low sedimentation coefficients compared with prothrombin and thrombin (237, 238). Although the generation of prothrombin fragments was discussed in two articles in this journal in 1954 (235,238), it has been only in the last 3-4 years that the activation products of prothrombin have been isolated and chemically characterized. Human prothrombin activation products that were electrophoretically (241), hydrodynamically (239), and chromatographically (10) distinct from either prothrombin or thrombin were described by both Lanchantin and co-workers (240, 239, 241) and Aronson and Menache (10, 11) in their studies in the mid-1960 ‘s, although these too were never characterized further. Since the amino acid sequences are available only for bovine prothrombin activation products, these are described in detail, even though the original identification of the prothrombin products was made on human material. The identification of two to three activation fragments that are formed from bovine prothrombin, depending on the activation conditions (see below), has been made independently in many laboratories (60,111,123,145,165-167, 199, 203, 283, 284, 289, 303, 309, 322, 326-328, 359, 425, 435, 443, 470). In addition to these products and thrombin, two transient species or “intermediates” are observed when sodium dodecyl sulfate (SDS) electrophoresis is used to monitor the activation process. In the study of the products of prothrombin activation by Owen et al. (359) and Heldebrant et al. (165, 167) composition and molecular weight were obtained from a variety of independent techniques so that the entire mass of the prothrombin molecule could be accounted for in the two usually observed activation fragments (plus a fragment equal to the sum of these two) and thrombin. These studies eliminated the ambiguity that existed in the previous work on bovine prothrombin proteolysis products (123, 166). Detailed chemical and physical-chemical characterization of all the isolated bovine prothrombin proteolysis products has now been reported by several laboratories. Data on molecular weight obtained from the many studies on bovine and human prothrombins and their activation products are summarized in Table 1. The general structure of the prothrombin



Identity

Product

Determination

KCl,

S/D

Bovine thrombin

Human Prethrombin 2

Bovine Prethrombin 2

Human Prethrombin 1

160

(polypeptide

c’

(polypeptide

only)

only)

(polypeptide

only)

37,700 2 100 glycoprotein) r

35,404

37,700 2 100 (glycoprotein) *

35,404

(glycoprotein)

49,900, v = 0.71

bt50,400

48,143

275, 276

275, 276, 303

275, 276 sed eq

V = 0.726

KC1 (33,600, v = 0.69), S/D

38,200',

6M Gu HCl, sed eq

37,000, V = 0.725,

sed eq

39,500, V = 0.718,

HCl,

159

285, 359

165

36,000-41,00@

30,000-35,000h

41,000'

37,000-40,000"

51,oool

57,000-65,000"

50,400 (46,200-52,400) v = 0.72; 6 M Gu

Bovine Prethrombin 1

165, 359, 493

67,800, 70,000'

sed eq

sed eq

495

NaPO,,

Gu HCl,

72,000"

275, 276

229

only)

72,000 2 3,000, 9 = 0.71, NaCl 83,560, V = 0.72; 74,000', v = 0.70', 6 M Gu HCl

(polypeptide

72,600 + 700 (glycoproteii$

66,098

Human prothrombin

Bovine prothrombin

72,000"(72,00084,000)

SDS gel electrophoresis

75, 159, 237

Weight

70,000 (67,000-68,500) 9 = 0.71, S/D

13. Mech anism for prothrombin l-2 must be part of the kinetic

.PRETHROMBIN ..... ..... .... .... .... .... .... .... .2... FRAGMENT 1.2 activation. intermediate

,A> I

THROMBIN + FRAGMENT

la2

Both Prethrombin 2 and Prothrombin in prothrombin conversion to thrombin.


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discussed by Magnusson (275, 276) and extensively by Silverberg and Nemerson (453) and Nemerson et al. (346). The requirement for positive thrombin feedback to “activate” Factor V (46, 83, 85, 86, 117, 263, 264;391, 393,418) and either thrombin (46, 83,85, 86, 117, 263, 264, 364, 391, 393, 418) or Factor Xa to activate Factor VIII (84) are also compatible with the notion of localized control reactions, as is the apparent stoichiometric relationship between Factor V and thrombin in Factor V activation (105, 411), which may serve as a proportional control process. These positive-feedback reactions involving thrombin and Factor Xa in fact provide an additional, independent mechanism for generating thrombin virtually explosively, but still locally. Unless some quantities of Factor V and Factor VIII are always present in their “activated” forms, the thrombin involved in the “activation” of these factors (or Factor Xa for Factor VIII activation) must arise via an activation process that occurs without the full participation of these two protein accessory factors. From the preceding description of prothrombin activation, it is reasonable to speculate that formation of thrombin required for “activation” of Factors V and VIII may occur primarily and possibly exclusively as a consequence of the phospholipid acceleration of the clotting reactions. The normal physiological significance of the interaction of the prothrombin activation system proteins with phosphilipid surfaces is realized most easily when the fact that only the lipid bindingrelated aspect of the prothrombin activation process is defective when das-ycarboxyprothrombin (116) and/or putatively dasy-carboxy Factor Xa (92) are substituted for the normal forms of these proteins. One obvious implication is that oral anticoagulant therapy with vitamin K antagonist owes its effectiveness to its elimination of calcium-mediated association between vitamin Kdependent proteins and phospholipids and the contribution of these interactions to the coagulation process. It also is significant that the normal concentration of bovine prothrombin is about l-2 PM (10, 272,470), a concentration far in excess of the concentration of thrombin formed in clotting blood (47). As the high steady-state plasma prothrombin concentration therefore is unlikely to exist for the purpose of conversion completely to thrombin, it is interesting to ask why so much prothrombin exists in the normal steady state. Quantitative measurement of prothrombin binding to phospholipid bilayer surfaces indicates that the concentration for half-saturation of bilayers containing an equimolar mixture of phosphatidylcholine and phosphatidylglycerol at l-2 mM Ca2+ (Dombrose, Gitel, and Jackson, unpublished observati .ons) is very nearly equal to the concentra tion of circulating prothrombin. Therefore th .e high prothrombin concentration may be maintained in order to ensure its association with lipid when the phospholipid membrane surface becomes accessible, such as might occur upon injury. Although it is not within the scope of this review to consider platelet participation in blood coagulati .on and hemostasis, it is we1 .I established that the hemostatic process involves not only plasma coagulation factors but platelets as well, and under many circumstances platelets can provide the phospholipid that participates in the coagulation (365, 410, 506, 507) and


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prothrombin activation processes (209). Moreover, a number of the components of the coagulation cascade are known to be associated wi .th platelets (51, 52, 56, 209, 365, 488), suggesting another mechanism by which the coagulation components are localized as a result of platelet adhesion to the newly exposed cell and tissue surface at sites of injury. III.

BIOSYNTHESIS

OF

PROTHROMBIN

Knowledge of the cellular events responsible for the production of prothrombin and the regulation of these metabolic events has come largely from investigations spanning only the last 10 years. In the mid 1930’s, vitamin K was discovered by Dam (79) as a dietary antihemorrhagic factor needed to maintain plasma levels of prothrombin; by the late 1930’s and early 1940’s, Link’s laboratory had identified the coumarins (61) as indirect anticoagulants that functioned by antagonizing the action of the vitamin (Fig. 14). During the subsequent 25 years a great deal was learned about the biological activity of various forms of both the vitamin and its antagonists and about the significance of the vitamin in animal nutrition and human medicine. Although Andersen and Barnhart (4, 5) conclusively demonstrated that prothrombin was produced in the liver, the lack of a general understanding of the mechanism of protein biosynthesis prevented serious experimental approaches to the cellular or molecular mechanisms involved until the last decade. As an example of the rate of advance in this field, the proceedings of a major symposium held in honor of Professor Dam in 1966, entitled “Recent Advances in Research on Vitamin K and Related Quinones,” devoted only a few pages (299) to a discussion of the mode of action of vitamin K in animals.

Warfarin [ 3-(

wacetonylbenzyl) hydroxycoumarin

Vitamin (2FIG.

methyl-3-

14. Structure

phytyl-

of vitamin

-4-

1

- K, I, 4 -naphthoquinone) K, (phylloquinone)

and Warfarin.


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However, progress has been rapid since the mid 1960’s and a number of recent reviews of the mechanism of action of the vitamin (355,356,469,479,480,484, 521) are now available. The general field of vitamin K metabolism has been reviewed periodically since its discovery. Almquist (3) and Dam (80) reviewed this field shortly after the discovery of the vitamin, and then somewhat later Dam (81) and Isler and Wiss (195) surveyed the vitamin K literature. The chemistry, metabolism, and nutritional aspects of the vitamin have been adequately reviewed and are not discussed here. Serious studies of the mechanism of prothrombin biosynthesis were begun about 10 years ago, and investigations over the last few years have tended to rule out many possible mechanisms of synthesis and have indicated that prothrombin is formed in the liver by a vitamin K-dependent carboxylation of a liver precursor protein.

A. Indirect

Evidence

for a Prothrombin

Precursor

Protein

The possibility that a precursor protein was involved in the formation of prothrombin was probably first clearly stated by Hemker et al. (177), who were studying the coagulation of plasma from patients receiving anticoagulant therapy. The time course of prothrombin appearance in the plasma observed by a number of investigators (39,183,212,384,477) when vitamin K was administered to severely hypoprothrombinemic rats was also consistent with the presence of a significant pool of a precursor protein that could be converted to prothrombin after vitamin administration. There was a delay in the appearance of plasma prothrombin that usually lasted 30-60 min after vitamin K administration, followed by a burst of synthesis. The time required for synthesis to begin was somewhat dependent on whether the hypoprothrombinemia was produced by coumarin administration (antagonism of vitamin action) or by a nutritional deficiency of the vitamin and was also influenced by the dose of vitamin used. Both Pyorala (384) and Bell and Matschiner (39) have clearly pointed out that the rate of prothrombin synthesis observed during this initial period exceeded the theoretical induction curve based on the experimentally determined half-life of prothrombin. Dulock and Kolmen (97) also observed a rapid increase in plasma prothrombin when vitamin K was administered to dogs previously given Warfarin, and their data again suggested the finalization of a liver precursor. The possible existence of a precursor was strengthened when it was shown (445) that the appearance of plasma prothrombin was preceded by a transient increase of prothrombin in rat liver microsomal preparations. Microsomal prothrombin peaked about 10 min after vitamin K was administered to hypoprothrombinemic rats and then fell as prothrombin appeared in the plasma. This response strongly suggested that in the hypoprothrombinemic rat a pool of precursor was present that could be converted to prothrombin in a vitamindependent step and that, after depletion of this pool, the rate of synthesis would slow and become dependent on the rate of synthesis of this precursor.


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It was demonstrated (39, 183, 477) that the vitamin K-stimulated initial burst of prothrombin in the hypoprothrombinemic intact rat was decreased only slightly by prior administration of the protein synthesis inhibitor cycloheximide. The amount of cycloheximide used in those experiments, however, was shown to block prothrombin synthesis when injected into a normal rat (39, 376); it was also shown (477) that the increase in plasma prothrombin observed between the 1st and 2nd h after vitamin K administration is blocked by this dose of cycloheximide. These studies, which utilized inhibitors of protein biosynthesis in intact animals, strongly suggested that protein synthesis was not involved in the vitamin K-dependent step of prothrombin synthesis, but did not offer final proof. It was always possible that, although the amount of inhibitor used was sufficient to block the synthesis of most proteins in the system studied, it was for some reason not blocking the formation of prothrombin. More conclusive and direct evidence of the presence of a liver precursor protein was obtained when Shah and Suttie (444) demonstrated that the prothrombin produced when hypoprothrombinemic rats were given vitamin K and cycloheximide was not radioactively labeled if radioactive amino acids were administered at the same time as the vitamin. These data strongly suggested that plasma prothrombin must have been derived from an existing precursor pool. If the vitamin had initiated de novo synthesis of prothrombin, and for some reason prothrombin synthesis was not blocked by cycloheximide, the newly formed prothrombin should have contained a high level of radioactivity. This study further indicated that administration of radioactive amino acids to hypoprothrombinemic vitamin K deficient rats prior to cycloheximide and vitamin K administration resulted in the formation of radioactive plasma prothrombin. This observation was consistent with the presence of a precursor protein pool that was being synthesized rapidly and could be converted to prothrombin in a step that did not require protein synthesis. Similar data were subsequently obtained by Olson et al. (356), who explained the decreased specific radioactivity of the prothrombin formed by postulating the presence of a specific peptide acting as a reserve protein pool that could specifically be broken down to furnish amino acids for prothrombin synthesis. No additional support for this theory has been provided. Although the observations in intact animals have rather consistently supported the existence of a precursor to prothrombin, experiments in perfused livers have been less conclusive. Puromycin has been reported to be both effective (476) and ineffective (353) in blocking a clotting factor response tc the vitamin, and Kipfer and Olson (228) have reported that in an isolated perfused liver vitamin K is able to specifically reverse the effect of cycloheximide on those ribosomes synthesizing prothrombin but not on the general ribosomal population. Some of the confusing data might be related to observations (355) that vitamin K administration in the perfused liver results in excretion of incompleted forms of prothrombin that do not have biological activity. As plasma clotting Factors VII, IX, and X also depend on vitamin K for their synthesis, studies of their formation should also contribute to an


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understanding of the mechanism of prothrombin synthesis. Studies of Factor VII formation in liver slices or isolated liver cells (14, 257, 377, 380, 390) have not contributed a great deal to an understanding of the system. More recent studies (15, 258, 382, 398) of Factor VII formation in cell-free systems have also been inconclusive but have tended to support the hypothesis that there is a liver precursor to Factor VII that can be converted to Factor VII without the need of additional protein synthesis. The results of a number of indirect studies of the action of vitamin K in promoting prothrombin synthesis during the late 1960’s and early 1970’s therefore tended to rather strongly support the hypothesis that prothrombin was being synthesized from some precursor protein that would pile up in the liver of vitamin K-deficient animals and could be converted to prothrombin in a vitamin K-dependent reaction not requiring protein synthesis. None of these data, however, could be used to conclusively establish such a route. B. Immunochemical

Evidence for a Prothrombin

Precursor

The hypothesis of a liver precursor to prothrombin was strengthened by observations that the plasma of man or animals treated with coumarin anticoagulants contained a protein similar to prothrombin in many ways. Hemker et al. (177) first postulated the existence of such a protein in the plasma of human patients receiving anticoagulant therapy by indirect means. Later, a protein antigenetically similar to prothrombin but lacking biological activity was demonstrated in such plasma by a number of workers (58,63,88, 137, 175,217,218,349). A similar protein was first demonstrated in bovine plasma by Stenflo (460), and its existence was subsequently confirmed by others (282, 342, 395, 505). The presence of such a protein in the plasma of other species administered oral anticoagulants has been somewhat controversial, and evidence for its existence has been sought by both immunochemical methods and by the assay of thrombin generation by nonphysiological activators. There are reports (213, 372) that plasma from rats administered anticoagulants does contain an appreciable amount of an abnormal form of prothrombin, but other investigators (62, 355) have found very little of this species. Both Olson (355) and Carlisle et al. (62) observed some abnormal prothrombin in the plasma of anticoagulant-treated chicks; Carlisle et al. (62) also reported that this protein appeared to be missing in plasma from anticoagulantitreated mice, hamsters, guinea pigs, rabbits, and dogs. In addition to the reports of immunochemically similar but biologically inactive forms of prothrombin in plasma after anticoagulant treatment, there is evidence of similar proteins corresponding to the other vitamin K-dependent clotting factors. The presence of an inactive form of Factor X has also been postulated on theoretical grounds (174). It has been reported that Factor X (88, 138, 383) and Factor IX (88, 246, 247, 503) antigenic-reacting material is found in the plasma of human patients treated with anticoagulants. The existence of such an abnormal protein with antigenic determinants for Factor VII is less clear. Evidence for such a protein has been reported (88, 151), but


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Prydz (381) failed to note the presence of this protein in an earlier study of a Factor VII antiserum. More recently, Howarth et al. (190) found such a protein in the majority of patients they studied. Reekers et al. (395) reported the presence of abnormal Factor IX and X as well as abnormal prothrombin in the plasma of cattle treated with a vitamin K antagonist. C. Isolation

and Characterization

of Abnormal

Prothrombin

The identification of these new proteins in the plasma of patients on anticoagulant therapy provided the stimulus for a series of investigations that culminated in an understanding of the chemical nature of the postribosoma1 modification of prothrombin. The protein from human plasma has been purified (63,454) but has not been subjected to extensive chemical characterization. The protein from bovine plasma has been purified in the laboratories of Stenflo (467), Suttie (342), Malhotra (281), Hemker (176), and Prydz (505); the majority of the information regarding its structure, however, was obtained by the first two groups. This protein has been given different names by various investigators: protein induced by vitamin K absence (PIVKA), abnormal prothrombin, isoprothrombin, Dicumarolized prothrombin, Dicumarolinduced prothrombin, or atypical prothrombin. Although perhaps each name has some merit, the plasma protein produced in the bovine administered an oral anticoagulant is called “abnormal prothrombin” in subsequent discussions. The initial studies of this protein (342, 462, 467) indicated that it appeared to have the same molecular weight and amino acid composition as normal prothrombin and that it did not adsorb to insoluble barium salts as did normal prothrombin. The lack of barium salt adsorption and the calciumdependent electrophoretic and immunochemical properties (462, 467) suggested a difference in calcium-binding properties of these two proteins that was directly demonstrated by Nelsestuen and Suttie (340) and confirmed by Stenflo and Ganrot (468). The difference in calcium binding was shown by Stenflo (461) to be a property of an amino-terminal peptide (Prothrombin Fragment 1) that could be derived from the two proteins. The observation (342) that the abnormal prothrombin could yield thrombin when treated with trypsin or snake venoms containing prothrombin activators indicated that this portion of the molecule was normal and that the critical difference in the two proteins was the inability of the abnormal protein to bind to calcium ions that are needed for the phospholipid-stimulated activation by Factor Xa. Although the phospholipid interactions were not investigated during the early studies of the abnormal prothrombin, they have now been directly probed. It has been shown (116), as was previously postulated, that the abnormal prothrombin will not bind to a phospholipid surface in the presence of calcium ions and that the addition of phospholipid, which drastically stimulates the Xa-Ca2+ activation of prothrombin, has no effect on the rate of activation of abnormal prothrombin. Although it was suggested (463) that anomalous pairing of disulfides might account for these differences, other


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observations (340) made this unlikely. A study (48) of the conformation of these two proteins by optical rotatory dispersion and circular dichroism revealed that they were indistinguishable in the absence of calcium ion, but that spectral changes occurred in normal prothrombin, on addition of calcium ions, that were not seen in the abnormal prothrombin. The early studies (342, 462, 467) of abnormal prothrombin indicated that its carbohydrate content and structure were probably similar to those of normal prothrombin (341) and offered final refutation of earlier claims (214, 372) that the vitamin K-dependent step in the formation of prothrombin involved glycosylation of the protein. Other metabolic studies (373) and the observation (178, 339) that asialo- and aglycoprothrombin retain biological activity and still adsorb to barium salts also made this hypothesis unlikely. Although it has more recently been claimed (331) that the abnormal prothrombin from human plasma does differ from normal plasma prothrombin in its carbohydrate content, this has not been confirmed; if so, it would differ appreciably from the major species of bovine abnormal prothrombin that has been characterized. There may be other minor species in the plasma, however, and it seems clear from a series of studies by Malhotra and Carter (279282) that different preparations of abnormal bovine prothrombin may be obtained from plasma, depending on the initial steps in the isolation, and what has been purified by one group may have been thrown away in an early purification step by another. The initial studies of the abnormal prothrombin clearly implicated the calcium-binding region of prothrombin as the vitamin K-dependent region, but provided no evidence of the chemical nature of this region. This problem was directly approached when Nelsestuen and Suttie (343) isolated an acidic peptide from a tryptic digest of normal bovine prothrombin that would adsorb to insoluble barium salts and bound calcium ions in solution. This peptide, which was a portion of the Fragment 1 region of prothrombin (see Fig. 2), contained a high proportion of acidic amino acid residues and had an anomalously high apparent molecular weight on molecular-sieve columns. Stenflo (463) later isolated two acidic peptides from the methods, and both groups postulated Fragment 1 region by different the existence of some unknown acidic, nonpeptide, prosthetic group attached to this portion of the molecule. The peptides isolated by these two groups could not be obtained when similar isolation procedu.res were applied to preparations of abnormal prothrombin. Barium salt adsorption to abnormally acidic peptides have also been noted in the Fragment 1 portion of prothrombin by other investigators (40, 274, 454). The original tryptic peptide has been further degraded and studied (188) and a similar peptide was obtained from Factor X (189). D. Characterization

of y-Carboxyglutamic

Acid

Although all the investigators involved in attempts to characterize the calcium- binding portion of prothrombin were Cl .early looking for some acidic presumably group(s) attached to the gl utamic acid residu .es of prothrombin,


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through an ester linkage, the chemical modification was much simpler. Stenflo et al. (465) succeeded in isolating a tetrapeptide (residues 6-9 of prothrombin) that had an apparent sequence of Leu-Glu-Glu-Val and demonstrating by a combination of mass fragmentation, nuclear magnetic resonance (NMR) spectra, and chemical synthesis that the glutamic acid residues of this peptide were modified so that they were present as y-carboxyglutamic acid &amino-l, 1,3-propanetricarboxylic acid) residues (Fig. 9). Independently, Nelsestuen et al. (344), by rather similar methods, characterized ycarboxyglutamic acid from a dipeptide (residues 33 and 34 of prothrombin) that appeared originally to be Glu-Ser. An alternate chemical method of identification of these residues has also been published (526). These characterizations of the modified glutamic acid residues in prothrombin were confirmed by Magnusson et al. (278), who have shown that all 10 of the first 33 Glu residues in prothrombin are modified in this fashion. Further details of these characterizations have been published (126,329) and methods for chemical synthesis of this new amino acid have been reported (126, 330). Factor X is also a calcium-binding vitamin K-dependent clotting factor. The amino-terminal region of the light chain of Factor X is homologous with the amino-terminal region of prothrombin, and it has been shown (103,189) to also contain these modified glutamic acid residues. The amino-terminal region of Factor IX has a considerable amount of homology with prothrombin and Factor X (129), and it has been shown (469) to contain these vitamin Kdependent modifications. E. Isolation

of Rat Liver Prothrombin

Precursor

Proteins

One of the more significant observations in the studies of the abnormal bovine prothrombins was that, although they were activated at a very slow rate by physiological activators, it was possible to rapidly generate a thrombinlike activity from them by limited trypsin digestion or by treatment with Echis carinatus or Dispholidus typhus venom. This suggested that, if the concentration of the hypothesized liver precursor built up in the liver by hypoprothrombinemic animals, it might be detected by the release of thrombin after incubation with these snake venoms. Suttie (478) then demonstrated that thrombin activity was generated when microsomes were isolated from Warfarin-treated rats, solubilized with detergent, and the extract treated with Echis carinatus venom. A similar increase in this activity was seen when rats were made vitamin K deficient or injected with a second anticoagulant, 2-chloro-3-phytyl-1,4naphthoquinone (256, 446). The protein responsible for this activity (“precursor”) increased rapidly when Warfarin was administered and more slowly when the rats were placed on a vitamin Kdeficient diet. Further study (448) demonstrated that the amount of this microsomal precursor decreased rapidly when vitamin K was injected and, as its level fell, the amount of microsomal prothrombin increased and then fell as it moved out of the liver into the plasma. A protein has now been isolated (106) from the liver of Warfarin-treated


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rats that has the properties predicted for this precursor. The purified precursor is a glycoprotein that is both immunochemically similar to prothrombin and has a molecular weight indistinguishable from rat prothrombin. Both electrophoretic and isoelectric-focusing analyses indicate that the precursor is less negatively charged than prothrombin. Specific proteolysis of the precursor by thrombin, taipan snake venom, or clotting Factor Xa yielded fragments indistinguishable from those formed by similar proteolysis of prothrombin. This protein does not adsorb to BaSO, and its rate of activation to thrombin by Factor Xa and Ca2+ was not stimulated by the addition of phospholipid. This protein appears to be identical to prothrombin except that it does not contain sialic acid residues and does not contain y-carboxyglutamic acid. This protein has an isoelectric point (PI) of 5.8, and more recently (152) a second protein with properties very similar, but with a p1 of 7.2, has been isolated from the same microsomal preparations. The increased basic nature of this protein is a property of the amino-terminal region of the molecule, but the chemical alteration responsible for the shift in p1 has not been determined. It has not been determined which of these proteins is the physiological precursor of prothrombin (153). It has been shown that there is a proalbumin form of serum albumin in rat liver microsomes (219, 406) that contains a basic amino-terminal peptide that is cleaved before secretion, and a more basic protein might then be a more logical candidate for the true prothrombin precursor. It has also been shown (459, 516) that some microsoma1 proteins contain a rather large amino-terminal hydrophobic peptide region that presumably has a functional role in membrane attachment and localization. It therefore is possible that the true precursor of prothrombin synthesis might be substantially different from the proteins that have been purified. Precursorlike activity can be found (62) in liver microsomal preparations of species other than the rat, but none of these activities have been purified. Morrissey et al. (332) have isolated a protein from liver microsomes of Warfarin-treated rats with properties similar to that isolated by Esmon et al. (106), which they called isoprothrombin. This protein was obtained from an antibody affinity column; however, sufficient amounts of it have not been available to determine its chemical properties. F. In Vitro Prothrombin

Production-

Carboxylation

Final proof of the precursor theory of prothrombin production depends not only on the isolation of a protein with the properties predicted for a precursor but on the demonstration of the conversion of this protein to prothrombin in a defined system. Progress in developing suitable in vitro systems has been slow. A number of in vitro cell-free systems that did show an increase in Factor VII (15, 258, 382, 398) or Factor X (66) activity upon incubation have been described. Only one of these systems (258) responded to the in vitro addition of the vitamin and therefore they have had limited value in determining the requirements involved in the conversion of liver precur-


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sors for these clotting factors to the active factors. Johnston and Olson (215, 216) have described an in vitro system that produces radioimmunologically detectable amounts of prothrombin upon incubation. The amount of prothrombin produced in this system depends on the prior vitamin K status of the rats used, but does not respond to the in vitro addition of the vitamin. More recently, the synthesis of an immunoreactive prothrombin species has been demonstrated in a heterologous system with rat liver mRNA and rabbit reticulocytes (337). The material produced can be degraded by E. carinatus venom and appears to have a molecular weight of about 75,000. Presumably this material corresponds to the primary ge ne product (precursor protein) rather than to prothrombin, but it has not yet been completely characterized. The first vitamin K-dependent in vitro system that produced prothrombin was described by Shah and Suttie (447). In this sytem, postmitochondrial supernates from vitamin K-deficient rats were shown to respond to the addition of vitamin K by producing a significant amount of prothrombin as assayed by the standard two-stage assay. Prothrombin production was dependent on 0, and an energy supply and was inhibited by antagonists of vitamin K but not by cycloheximide. After the vitamin K-dependent step in prothrombin synthesis was shown to be the formation of y-carboxyglutamic acid residues, the same system was used to demonstrate (114, 482) that the addition of vitamin K and H14C0,- promoted the carboxylation of microsomal proteins (Fig. 15). This carboxylation had essentia .llY the same requirements as the in vitro prothrombin-synthesizing system. It was possible to isolate radioactive prothrombin from this system after incubation and show that essentially all the incorporated radioactivity was in the Fragment 1 region of prothrombin and that acid hydrolyses of this fragment resulted in the loss of 50% of the radioactivity. This was consistent with the acid lability of ycarboxyglutamic acid residues, which are chemically modified malonic acid derivatives. The radioactivity remaining after acid hydrolysis was exclusively present in glutamic acid. These observations would appear to offer final proof of the role of the vitamin in the biosynthesis process. An in vivo demonstration of H14CO:,- incorporation into prothrombin has also been reported (143), but although this reaction undoubtedly does occur the evidence presented regarding the specificity of the labeling was not conclusive. The vitamin K-dependent carboxylase has been studied (128, 144,407) in washed microsomes where the activity requires the presence of the precursor, 02, vitamin K, and HCO;{- and is stimulated by an energy source and Precursor

Prot h rombin

glutamyl residues residues FIG. 15. Vitamin K-dependent carboxylase. that converts peptide-bound glutamyl residues carboxyglutamyl residues in prothrombin.

Vitamin K is a required in microsomal precursors

cofactor in reaction of prothrombin to y-


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factor(s) present in the postmicrosomal supernatant. A major factor in the supernatant is a protein(s) acting as a NAD+ (NADP+) reductase. This requirement can be replaced with the addition of NADH or NADPH to the system, and the requirement for reducing equivalents from pyridine nucleotides in the systems has now been shown to be largely a requirement for the reduced form of vitamin K. It has also been shown (128) that dithiothreitol (DTT) can b e used as the source of reducing equivalents for this reaction and that this reducing agent might also be functioning to protect an essential sulfhydryl group in the enzyme system. The carboxylase activity in this microsomal preparation can be inhibited by Warfarin, and this inhibition can be overcome by high concentrations of the vitamin (407). The vitamin K-dependent carboxylase activity has now been solubilized in various detergents (115, 144, 267), and the solubilized preparation retains many of the properties of the membrane-associated system. The solubilized system is still stimulated by DTT and inhibited by mercuricals (267). It has been reported that the solubilized system is inhibited by the spin-trapping agent 5,5-dimethyl-1-pyroline-N-oxide and that 0, is not required in the solubilized system when the reduced form of the vitamin is used (144). The latter finding is, however, not supported by other data (115) showing a requirement for 0, in such a system. In contrast to the microsomal system, the solubilized system is not inhibited by Warfarin but is still sensitive to a direct vitamin K antagonist such as 2-chloro-3-phytyl-1,4-naphthoquinone (115). The solubilized system has been particularly useful in clarifying the need for ATP in the system. Incubation in the absence of ATP and the presence of an ATP inhibitor AMPP(NH)P does inhibit the membrane-bound (microsomal) carboxylase, suggesting that the energy to drive the carboxylation may come from the reoxidation of the reduced vitamin in the system, but the mechanism remains unclear. Studies of the mechanism should be facilitated by the observation of Suttie et al. (483) that the pentapeptide Phe-LeuGlu-Glu-Val will serve as a substrate for the carboxylase. G. Molecular

Function

of Vitamin

K

Although in one sense the efforts of the past few years have been successful in determining the mechanism of action of vitamin K, in another sense they have only opened up the field. It is clear that the vitamin functions to promote the carboxylation of a microsomal precursor to prothrombin, but there are essentially no data to establish what its role is in this reaction. Apparently there are three generalized possibilities: it may function to activate (or transfer) HCO,-(CO,) for this carboxylation, it may function to labilize the hydrogen at the y-carboxyl of the precursor so that it may accept the HCOJCO,) or it may function as an activator of one of the enzymes in this reaction. The last possibility is probably least likely. It would seem that a low-molecular-weight, biologically active substance with a redox potential in the physiological range would make use of its redox properties in its action and the observation that the hydroquinone, which is not the physiologically


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stable form, is the biologically active form would tend to substantiate this. At the present time there is little information to indicate which of the other two possibilities is most likely. Although it was originally assumed that ycarboxyglutamic acid residues were found only in plasma proteins produced in the liver, the demonstration of a y-carboxyglutamic acid-containing protein(s) in bone (163, 379) and renal tissue (162) indicates that the vitamin Kdependent carboxylase may be more widespread. Vitamin K-dependent carboxylation has been demonstrated in kidney cortex (162), but the product has not been identified. Any theory of the mechanism of action of vitamin K must take into consideration the possibility that the formation of vitamin epoxide is involved in the reaction. There has been an extensive investigation of the metabolism of the 2,3-epoxide of vitamin K (K-oxide) over the past few years (Fig. 16). Bell and Matschiner (37, 38) demonstrated that Warfarin administration blocks the action of a liver enzyme that reduces vitamin K-oxide to the vitamin so that during anticoagulation treatment there is a high ratio of Koxide to vitamin K in the liver. They postulated that vitamin K-oxide is a competitive inhibitor of the action of the vitamin and that Warfarin exerts its anticoagulant effect through a buildup of this metabolite, which then blocks the action of the vitamin. This theory has now been shown (150, 408) to be untenable. More recently, Willingham and Matschiner (518) postulated that the formation of the epoxide (“epoxidase” activity) is an obligatory step in the action of the vitamin in promoting prothrombin biosynthesis. This hypothesis was originally based on observations that “epoxidase” activity increased in liver under various treatment in much the same manner as concentrations of the prothrombin precursor. The theory is also supported by observations on the effects of various anticoagulants in normal and Warfarin-resistant rats (36, 517) and by observations that the requirements for in vitro epoxidation and vitamin K-dependent carboxylation are similar. The available evidence is far from conclusive, but it does suggest that both reactions involve some components of the microsomal electron transport system and that they might somehow be coupled.

0 (1

C

/

I

\ a

R

cc( Vitamin

0

C

0

I

R

0

0

K

Vi tamin epoxid

K e

Warforin FIG. 16. Vitamin K epoxide metabolism. Liver microsomes contain an enzyme system that will form the 2,3-epoxide of the vitamin and a second system that will reduce the epoxide to the vitamin. The second activity is strongly inhibited by coumarin anticoagulants.


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The molecular function of the vitamin therefore has not been established. It does appear, however, that a biochemical system is now available that should eventually yield the type of information needed to establish its role in prothrombin synthesis. There is also a considerable amount of disagreement (481) regarding the mechanism by which coumarin anticoagulants inhibit the action of vitamin K, and this system should also serve as a tool to investigate these interactions. H. Normal

Regulation

of Prothrombin

Concentration

Plasma prothrombin levels normally are held within a rather narrow range and the control that achieves this need not, and probably does not (23), involve the vitamin K status of the animal. It has been shown, for example, that albumin synthesis can be enhanced by hypoalbuminemia (403) and it is possible that prothrombin synthesis is subject to the same type of regulation. Liver microsomes isolated from hypoprothrombinemic rats that were previously deficient in vitamin K show an increased rate of prothrombin synthesis after vitamin administration (216). This increased rate is maintained until the plasma levels of prothrombin are restored to normal. Synthesis is decreased once normal levels of prothrombin are achieved, and this alteration in the biosynthetic rate certainly does not involve alterations in the tissue concentrations of the vitamin. The experimental design of these studies was such that what was being measured was probably an indication of the amount of prothrombin mRNA in the microsomal preparation. At present, there are no data available that would give any indication of how these levels might be regulated by plasma prothrombin concentrations, and this area remains an interesting problem for future investigations. Jason and Helgeland (204) have shown that prothrombin activity in normal rat liver is concentrated in smooth rather than rough microsomes. This approach and the development of a cultured liver cell system (334) that produces prothrombin should be useful tools in studying these regulatory events. I. Alternate

Theories

of Vitamin

K Action

A number of theories explaining the role of vitamin K in prothrombin synthesis have been proposed in the last 40 years. Dam et al. (82) originally proposed that the vitamin, or at least a portion of it, was a part of the prothrombin molecule, but this observation could never be confirmed. Martius postulated that the vitamin had a function in mammalian electron transport such that a deficiency would lead to a defect in oxidative phosphorylation and a low cellular ATP level. This would then result in a decrease in the concentration of a protein with such a rapid turnover rate as prothrombin. He cited evidence (300) of low P:O ratios in vitamin K-deficient chicks that were not supported by other investigators (366, 522). In the mid 1960’s, Olson (354) suggested that the rate of prothrombin production is regulated by


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an effect of vitamin K on DNA transcription. These observations were adequately refuted (183,257,476) and subsequent investigations centered around two alternate hypotheses: I) that the vitamin acts as a ribosomal site to regulate the de novo rate of prothrombin synthesis or 2) that it functions postribosomally in a metabolic step that converts a precursor protein, which can be produced in the absence of the vitamin, to active prothrombin. The previous discussion has followed the development of the precursor hypothesis to the point where it appears that this is the only tenable explanation of the available data. It was possible, however, to explain many of the experimental observations by either theory, and recent reviews by Olson (355, 356) trace the development of the de novo theory for control of prothrombin biosynthesis in his laboratory. The recent claim (301) that a vitamin K-dependent step in prothrombin synthesis involves a hydroxylation of an alanine to a serine residue also has not been confirmed.

IV.

CONCLUSIONS

In the past decade, but particularly in the last 5 years, our understanding of prothrombin chemistry, activation, and biosynthesis has advanced from a phenomenological level to the molecular level. Studies of prothrombin and Factor X chemistry have led to the elucidation of the primary structure of both prothrombin and its activating enzyme, Factor Xa. A comparison of the amino acid sequences of these two proteins shows both their structural similarities and the structural differences and provides a basis for more detailed investigation and understanding of how the specific functional properties of these proteins, and in particular their different specificities in the coagulation process, are related to their structure. The discovery of a new amino acid, y-carboxyglutamic acid, and the determination of the location of y-carboxyglutamic acid residues in the prothrombin amino acid sequence have opened a new area for protein chemical investigation and provided the basis for U .nderstanding the Ca2+-mediated associations that occur between the vitam .in K-dependent clotting factors and phospholipid membrane surfaces in the coagulation process. The physiological significance of such lipid-protein associations, which may be responsible for localizing the coagulation process at an injury site, are seen most easily from the demonstration that only the lipid-protein association aspect of the prothrombin activation process is defective when “abnormal” (dasy-carboxy) prothrombin replaces normal prothrombin in the prothrombin activation system. This latter observation has also provided a molecular explanation for the anticoagulant effect of the vitamin K antagonist drugs - viz., elimination of Ca2+-mediated association with lipids by the vitamin K-dependent coagulation factors. Studies of the mechanism of prothrombin activation have demonstrated that thrombin formation occurs via a two-step process with the integrity of the activation intermediate maintained via a noncovalent association be-


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tween the immediate thrombin precursor (Prethrombin 2) and the “pro” portion of prothrombin (Fragment 1 2). The “pro” half of prothrombin, which is not part of the thrombin molecule, participates during prothrombin activation by mediating the association between prothrombin and the phospholipid surface via the Fragment 1 region and between prothrombin and Factor Va via the Fragment 2 region. Binding between prothrombin and Factor Va requires “activation” of Factor V (the circulating form of this coagulation factor) by either thrombin or an exogenous enzyme. This latter observation provides a mechanism for a proteolytic activation process that does not lead to formation of an active proteolytic enzyme as is most frequently the case in the coagulation cascade. The multiple association reactions among the prothrombin activation activation process SYStern components serve both to localize the prothrombin and to provide the means by which thrombin formation can occur almost explosively. Such organization of the prothrombin activation complex, which occurs as a result of the prothrombin-Factor Va-phospholipid-Factor Xa pairwise associations, also protects Factor Xa from inactivation by the plasma proteinase inhibitor Antithrombin III. This latter consquence of the associations among the prothrombin activation system components reinforces the conclusion proposed above on the basis of quite different observations that organization of the activator is undoubtedly of considerable physiological importance. During the last 5 years, the action of vitamin K has been conclusively established to occur postribosomally and to result in the carboxylation of glutamate residues present in the polypeptide chains of the vitamin Kdependent coagulation factors to yield y-carboxyglutamate containing proteins. Evidence that a precursor of prothrombin exists in rat liver, which can be isolated and carboxylated in vitro, has been derived from a variety of types of experimental approaches. The mechanism by which vitamin K participates in the protein carboxylase reaction has not yet been determined, although it has been established that the quinone vitamin must be reduced to the hydroquinone prior to its action. The specific mechanism by which the vitamin K antagonist drugs block the vitamin’s action and the role of the vitamin K epoxide in the carboxylase reaction also remain to be determined. Without doubt, however, the investigations of the next few years into this process will provide breakthrough and excitement to match the last decade in this area of research. l

The authors on this manuscript.

thank

Drs.

L. Glaser,

P. Majerus,

and D. Silbert

for reading

and commenting

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Choline inhibition of prothrombin activation.

Rat prothrombin: purification, characterization, and activation.

Prothrombin structure: unanticipated features and opportunities.

Wool structure and biosynthesis.

Ferritin structure and biosynthesis.

Prothrombin activation induced by Ecarin - a prothrombin converting enzyme from Echis carinatus venom.

Dual effect of histone H4 on prothrombin activation.

Distribution of prothrombin carbohydrate units upon thrombin activation.

Binding of the products of prothrombin activation to human platelets.

Sterol molecule: structure, biosynthesis, and function.

Cytochrome f: Structure, function and biosynthesis.

Marine nucleosides: structure, bioactivity, synthesis and biosynthesis.

Structure and biosynthesis of nerve growth factor.

Prothrombin.

Decarboxylation of bovine prothrombin fragment 1 and prothrombin.

Precursor activation in a pyoverdine biosynthesis.

Allergen-dependent oxidant formation requires purinoceptor activation of ADAM 10 and prothrombin.

Inhibition of factor X, factor V and prothrombin activation by the bis(lactobionic acid amide) LW10082.

or carboxyl groups.

Immunological properties of canine prothrombin, its activation products, plasma and serum.

Investigation of the aggregation and activation of prothrombin using quasi-elastic light scattering.

Synthesis and proof of structure of the new amino acid in prothrombin.

The Ca2+ ion and membrane binding structure of the Gla domain of Ca-prothrombin fragment 1.

Inferior vena caval filters and systemic prothrombin activation in orally anticoagulated patients.