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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:754-758.)
© 1995 American Heart Association, Inc.

Articles

Evidence That Meizothrombin Is an Intermediate Product in the Clotting of Whole Blood

Edwin G. Bovill; Russell P. Tracy; Timothy E. Hayes; Richard J. Jenny; Francis H. Bhushan; Kenneth G. Mann

From the Departments of Pathology (E.G.B., R.P.T., T.E.H., F.H.B.) and Biochemistry (R.J.J., K.G.M.), University of Vermont College of Medicine, Burlington.

Correspondence to Dr Edwin G. Bovill, Chairman, Department of Pathology, University of Vermont College of Medicine, Burlington, VT 05405.

	Abstract

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Abstract Meizothrombin is an intermediate that is produced during the conversion of prothrombin to thrombin in systems composed of purified factor Xa and factor Va that are quantitatively assembled on an anionic phospholipid surface. The biological significance of this intermediate has recently been challenged by the apparent absence of meizothrombin during clotting of sodium citrate–anticoagulated plasma. We analyzed the formation of thrombin during coagulation of nonanticoagulated, unchilled, minimally manipulated whole blood in glass tubes. The process was stopped at 0, 3, 5, and 7 minutes by the addition of biotinylated peptidyl chloromethylketone active-site labeling reagents. Plasma/serum was separated by centrifugation, and labeled species were extracted by immunoadsorption with a polyclonal anti-prothrombin antibody. The purified prothrombin-derived species were separated by SDS–polyacrylamide gradient gel electrophoresis and visualized on a chemiluminescent avidin blot. Meizothrombin appeared as an intermediate product of this reaction and persisted with some increase through the 7-minute time point. We also observed incorporation of the active-site label into a species of lower molecular weight consistent with the B₁ chain of ß- and/or -thrombin. These degraded forms of thrombin have not been previously demonstrated in a biologically relevant preparation. Our data clearly establish the generation of meizothrombin as an intermediate product of thrombin generation during whole-blood clotting. The data also represent the first experimental evidence for the generation of ß- and -thrombin in a biologically relevant environment and time scale.

Key Words: thrombosis • meizothrombin • coagulation • whole blood

	Introduction

Top Abstract Introduction Methods Results and Discussion References

 
-Thrombin can be derived from prothrombin by two pathways, each of which generates different intermediate products (Fig 1). In one pathway, human prothrombin is converted relatively slowly to -thrombin in the presence of factor Xa and Ca²⁺ ions by initial cleavage at residue (R) 271 (with consequent production of fragment 1.2 and prethrombin 2) followed by cleavage of prethrombin 2 at R 320 (with consequent generation of -thrombin).¹ In the other pathway the prothrombinase complex, consisting of the Ser protease factor Xa and the cofactor factor Va assembled on a phospholipid surface in the presence of Ca²⁺,^{2 3 4} cleaves the two bonds in reverse sequence with a five-order–magnitude increase in reaction rate. Initial cleavage at R 320 yields an intermediate product, meizothrombin, which is an active enzyme. Prothrombinase then cleaves meizothrombin at R 271 to yield -thrombin. Meizothrombin and thrombin can autocatalytically cleave at R 155, producing fragment 1 and the enzyme meizothrombin(desF1),⁵ and at R 284, producing an -thrombin–like product with a 13-residue deletion at the NH₂ terminal of the A chain (Fig 1).⁶

View larger version (28K): [in this window] [in a new window]   Figure 1. Diagrammatic representation of prothrombin activation products. Prothrombin and the prothrombinase-generated activation products are diagrammed. Prothrombin fragments 1 and 2 along with the A and B chains of thrombin are identified. Thrombin catalyzes cleavage at positions 155 and 284, whereas factor Xa catalyzes cleavage at positions 271 and 320. Cleavage sites associated with ß- and -thrombin are also identified, as are the pathways for human prothrombin activation. Prothrombinase-catalyzed cleavage occurs at positions 320 and then 271, producing fragment 1.2 and -thrombin as the final reaction products. F1.2 indicates prothrombin fragment 1.2; P2, prethrombin 2; B, B chain of thrombin; A, A chain of thrombin; F1.2.A, prothrombin fragment 1.2 A chain; S-S, disulfide bonds; and A', des(271-284) A chain of thrombin.

The presence of meizothrombin as an intermediate product in the prothrombinase conversion of prothrombin to -thrombin has been demonstrated primarily in purified systems.^{7 8 9 10 11 12 13 14 15 16} However, the physiological significance of these observations has been challenged by Tans et al,¹⁷ who failed to detect significant amounts of meizothrombin during clotting of sodium citrate–anticoagulated plasmas that had been activated with thromboplastin and partial thromboplastin preparations. In contrast to the results of Tans et al,¹⁷ Tijburg et al¹⁸ demonstrated the presence and persistence of meizothrombin during factor Xa–catalyzed prothrombin activation on endothelial cells.

Anticoagulated plasma is a potentially misleading model for in vivo processes because the cellular elements have been discarded and most free calcium has been bound to citrate. Nonanticoagulated whole blood that has not been centrifuged, frozen, thawed, recalcified, etc, represents a closer approximation of the environment in vivo. This study describes the incorporation of biotinylated peptidyl chloromethylketones into the active sites of prothrombin activation products during clotting of minimally manipulated, nonanticoagulated, unchilled whole blood.

	Methods

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Biotinyl-dicaproyl-D-Phe-Pro-Arg-chloromethylketone (bc-FPRck) and biotinyl-dicaproyl-D-Glu-Gly-Arg-chloromethylketone (bc-EGRck) were provided by Haematologic Technologies. Burro polyclonal anti-human prethrombin 1 was prepared and purified as previously described.¹⁸ Sepharose and all general chemicals were obtained from Sigma Chemical Co with the exception of 2-mercaptoethanol, which was obtained from Aldrich Chemical Co. Vectastain ABC kit was obtained from Vector Laboratories, and enhanced chemiluminescent reagents were obtained from Amersham Corp. Meizothrombin was prepared as previously described.⁶

This study was reviewed by the institutional review board at the University of Vermont, Burlington. Blood was drawn from eight volunteers by using a 19-gauge butterfly and plastic syringes (Becton Dickinson). A tourniquet was applied for initiation of phlebotomy and released on achieving free flow of blood into the syringe. The first 3 mL was discarded, and a fresh 10-mL plastic syringe was used to collect the blood sample. Immediately on completion of phlebotomy, 2 mL blood was directly transferred into each of five 12x75-mm glass test tubes in a 37°C water bath, where they were gently agitated. Coagulation was stopped at 0, 3, 4, 5, and 7 minutes by addition of a mixture of bc-FPRck and bc-EGRck to achieve final concentrations of 50 and 5 µmol/L, respectively. The 0-time-point sample was preloaded with the bc-FPRck/bc-EGRck solution. After addition of the biotinylated chloromethylketones, the blood was stored on melting ice for less than 1 hour until centrifugation at 1000g for 30 minutes. Plasma/serum was then removed and stored at -70°C. Preliminary experiments demonstrated that whole blood clotted between 3 and 5 minutes in the 12x75-mm glass tubes at 37°C.

The plasma/serum samples were then thawed at 37°C and processed at 4°C. Quantitative immunoadsorption was conducted with polyclonal anti–prethrombin 1/Sepharose 4B that had been coupled at a concentration of 3.5 mg/mL as previously described.¹⁹ The resin (50 µL) was equilibrated with 0.01 mol/L sodium phosphate–0.15 mol/L NaCl, pH 7.4 (PBS). Plasma/serum (50 µL) was added to 50 µL resin in a polypropylene microfuge tube (National Scientific Supply) and gently mixed on a Vortexer-2 (VWR) for 30 minutes at room temperature. The tubes were then centrifuged at 13 000g for 1 minute, and the supernatant was shown to be completely depleted of prothrombin (<0.01%) as assayed by a prothrombin enzyme-linked immunosorbent assay.²⁰ The resin was washed six times with 1 mL PBS and then eluted with 200 µL of electrophoresis sample prep buffer consisting of (vol/vol) 2% SDS and 1% CHES in 25% glycerol water. The elution buffer and resin were heated to 100°C for 5 minutes, allowed to cool to room temperature, and then centrifuged at 13 000g for 10 seconds at room temperature. The samples were divided into two parts, with one part constituted to 5% 2-mercaptoethanol, and then stored at -20°C.

Samples were subjected to electrophoretic analysis on a 7.4% to 15% SDS–polyacrylamide gradient (SDS-PAGE) gel and transferred to nitrocellulose as previously described.²¹ The nitrocellulose blots were then blocked with 5% milk powder and incubated with Vectastain ABC–immunoperoxidase and developed with Enhanced Chemiluminescence Reagent (Amersham Ltd) according to the manufacturer's directions. Once developed, the blots were exposed to X-OMAT RP x-ray film (Kodak Inc).

	Results and Discussion

Top Abstract Introduction Methods Results and Discussion References

 
The meizothrombin-standard preparation labeled with bc-FPRck (Fig 2) was subjected to 7.4% to 15% SDS-PAGE and transferred to nitrocellulose with subsequent analysis by avidin peroxidase. The results demonstrated incorporation of bc-FPRck into the appropriate product at M_r 70 000. A trace of meizothrombin(desF1) was also present at M_r 50 000. After the preparation was reduced with 2-mercaptoethanol, the biotin reagent appeared to have comigrated with the B chain of -thrombin, consistent with the specific labeling of the active-site location of the active-site His (Fig 1).

View larger version (29K): [in this window] [in a new window]   Figure 2. Avidin blot of the meizothrombin standard in the presence (right) and absence (left) of 2-mercaptoethanol. Molecular weights (x103) are displayed along the left-hand margin. mIIa indicates meizothrombin; mIIa desF1, meizothrombin des fragment 1; and IIa B chain, B chain of -thrombin.

Eight individuals were phlebotomized in this series of experiments. An avidin blot analysis following SDS-PAGE from a representative whole-blood clotting experiment is shown in Fig 3, with lanes 1, 2, and 3 representing 0, 3, and 7 minutes, respectively, after phlebotomy. Prothrombin and its derivatives were quantitatively extracted by immunochemical methods from these timed samples, eluted, and run on an SDS-PAGE system with subsequent transfer to nitrocellulose for avidin blotting. Lane 4 of Fig 3 is the meizothrombin- standard preparation. In this particular experiment, whole blood clotted between 3 and 5 minutes, times that were representative of all eight individual samples. Despite the slight variation in clotting time, the results for the individual experiment shown in Fig 3 were similar for all eight tested individuals. It is apparent from the nonreduced gel in Fig 3A that meizothrombin is present at the 3-minute time point and persists even after visible clot formation. Meizothrombin was observed after the 0 time point in all eight experiments. There is also a band at 7 minutes that comigrated with meizothrombin(desF1), along with a slightly lower-molecular-weight band that cannot be definitively identified from these data. Meizothrombin(desF1) was identified in 7 of 8 experiments, with the slightly lower-molecular-weight band present in 6 of 8 cases. It is also apparent that small amounts of -thrombin occur at the 0 time point. In fact, -thrombin was detected in 6 of 8 experiments at the 0 time point. On the nonreduced gel at all three time points, there are two bands just below thrombin. The larger of the two most likely represents -thrombin(des 272-284) as a consequence of thrombin cleavage at R 284.²² This product was present in all experiments. The smaller of the two bands represents an as yet unidentified proteolytic derivative of thrombin and was present in 3 of 8 experiments. The reduced gel in Fig 3B demonstrates the appearance of a band that comigrated with the B chain of -thrombin, as would be appropriate for reduced meizothrombin or meizothrombin(desF1). Also present on both the nonreduced and reduced gels at the 7-minute time point is a low-molecular-weight band at less than M_r 16 000, which contains the active-site His–bound biotinylated chloromethylketone. This faint band most likely represents the B₁ chain that results from the generation of ß- and/or -thrombin and was detected in 4 of 8 experiments.

View larger version (40K): [in this window] [in a new window]   Figure 3. Avidin blots of representative clotting experiment. Lanes 1, 2, and 3 are the 0-, 3-, and 7-minute time points, respectively. Lane 4 is the meizothrombin standard. A, Under nonreduced conditions; B, in the presence of 2-mercaptoethanol. The experimental protocol is described in "Methods."

The methods in the present study employ a double selection process. In the first step, active-site His (ie, His 363) are blocked and labeled with biotinylated peptidyl chloromethylketones. In the second step, prothrombin and its derivatives are extracted by quantitative immunoadsorption. Thrombin–antithrombin III and thrombin–heparin cofactor II complexes are not detected, because only His 363–labeled species are identified by the avidin-peroxidase detection system and the antithrombin-thrombin inhibitor complex blocks His 363. The His 363–labeled B chain of thrombin (Table 1) can be followed during and after clotting of nonanticoagulated whole blood, thus allowing observation of -thrombin generation and degradation.

View this table: [in this window] [in a new window]   Table 1. His 363 Products

The data presented herein provide evidence for the presence and persistence of meizothrombin as an intermediate product of prothrombin activation during clotting of nonanticoagulated whole blood. These findings are markedly different from those of Tans et al,¹⁷ who found little or no evidence of meizothrombin generation during clotting of sodium citrate–anticoagulated plasma and concluded that meizothrombin had no physiological significance. The lack of blood cells or the effect of sodium citrate anticoagulation may explain the absence of meizothrombin observed by these investigators. The clotting of nonanticoagulated, unchilled whole blood appears to be a closer approximation of in vivo clot formation than that of manipulated, anticoagulated plasma. We also observed a small amount of meizothrombin(des1), which is consistent with experience in purified systems.^{10 11 12 13 14 15 16 17 18}

-Thrombin plays a number of essential roles in the processes of blood coagulation. Thrombin converts fibrinogen to fibrin and activates factors V, VIII, XI, and XIII.^{23 24 25} Thrombin binds to the endothelial cell receptor thrombomodulin to activate protein C.^{26 27 28} Activated protein C is an anticoagulant that inactivates factors V and VIII, thus limiting thrombin generation.^{26 29 30 31} The -thrombin that is bound to thrombomodulin exhibits diminished activities toward substrates other than protein C.^{32 33} Thus, thrombin plays a role in modulating its own procoagulant activity. Thrombin also plays a role in modulating cellular events in coagulation, including activation of platelets,^{23 24 25} stimulation of prostacyclin release by endothelial cells,^{34 35} vasoconstriction,^{36 37 38} and growth factor activity.^{35 39 40 41} Therefore, study of -thrombin production mechanisms is of key importance in understanding coagulation and thrombosis.

The physiological role of meizothrombin is controversial. Previous work from our laboratory with purified, naturally occurring proteins demonstrated that meizothrombin did not recognize the major procoagulant substrates of -thrombin, including factor V, fibrinogen, and platelets.⁷ By contrast, meizothrombin activity toward protein C, which is similar to -thrombin in the presence of thrombomodulin and phospholipid surfaces, pointed to a possible regulatory role for meizothrombin in hemostasis. However, data from two other laboratories using recombinant proteins have shown results that conflict with ours. Wu et al⁹ were unable to demonstrate binding of recombinant meizothrombin, wherein the active-site Ser was replaced by Ala, to recombinant thrombomodulin expressed on the surface of CV-1 cells. In another recent report, Tans et al⁸ demonstrated a meizothrombin-induced factor V activation rate similar to that of -thrombin in a purified system constituted with recombinant human meizothrombin, in which the susceptible R 156 is replaced by Ala.⁸ The differences between natural and recombinant products need to be reconciled. Natural meizothrombin is inherently unstable owing to autoproteolysis, and recombinant proteins may not mirror the natural product because of differences in posttranslational processing, folding, etc.

An undisputed and potentially important role for meizothrombin is as a mediator of vascular constriction. Meizothrombin has potent vasoconstrictive activity, approximately fivefold greater than -thrombin.³⁸ This activity appears to be mediated by a direct effect on vascular smooth muscle.

In commercial preparations, -thrombin undergoes proteolysis, with the generation of ß-thrombin and -thrombin. These degraded forms of thrombin were first observed in bovine preparation by Mann and Batt⁴² and have not been reported in purified activation systems or in association with the clotting of whole blood or plasma. Fig 1 illustrates the derivation of these degraded forms of thrombin. Loss of clotting activity by ß-thrombin appears to be due to the cleavage of residues 63 through 73 in the B chain.⁴³ ß-Thrombin and -thrombin retain amidolytic activity toward small substrates but have markedly diminished proteolytic activity toward fibrinogen, factor V, and protein C and do not activate platelets.⁷ Our data are the first to suggest that ß- and -forms of thrombin can be generated in other than commercial thrombin preparations and trypsin-treated preparations of purified thrombin. These degraded forms of thrombin have not been previously described in biological preparations and, thus, not considered biologically relevant. -Thrombin can be stored in solution for months without evidence of "autolysis," whereas there is rapid production of degraded forms of thrombin in these experiments during clot formation. Thus, the precise enzymatic pathway that produces the degraded forms of thrombin is unclear. Because the proteolyzed forms of -thrombin have markedly diminished activity toward factor V, fibrinogen, and platelets,^{44 45 46} generation of these forms during clot formation may constitute a significant feedback loop in control of thrombin generation and activity. Together these data support the notion that a number of proteolytically active prothrombin activation products are formed at significant levels during prothrombin activation and may exist at significant concentrations in vivo.

It is of some interest that our data demonstrate the presence of -thrombin even in the 0-time-point sample in 6 of the 8 individuals tested. Whether this represents an artifact of phlebotomy or the presence of circulating thrombin in vivo cannot be determined from our experiments. Finally, we observed two bands on the avidin blot that migrated to positions that do not coincide with those of known B₁ chain species, one just below meizothrombin(desF1) and the other just below -thrombin (des 272-284). These may represent as yet undescribed intermediate and/or degradation products of thrombin.

In summary, this work clearly establishes that meizothrombin is present during the clotting of nonanticoagulated whole blood and therefore is important in evaluating its physiological significance. The presence of residual meizothrombin even after clot formation suggests there may be additional roles for this molecule in vascular function. Finally, these data represent the first experimental evidence for the generation of ß- and -thrombin in a biologically relevant environment and time scale.

	Acknowledgments

 
This study was supported by grants HL-46703 (to Dr Mann) and HD-24084 (to Dr Bovill) from the National Institutes of Health, Bethesda, Md.

Received November 21, 1994; accepted April 4, 1995.

	References

Top Abstract Introduction Methods Results and Discussion References

 

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