membrane binding by prothrombin mediates its constrained

Bradford, Orcutt & Krishnaswamy Version: 6 Aug 2013

Membrane Binding by Prothrombin Mediates its Constrained Presentation to

Prothrombinase For Cleavage.

Harlan N. Bradford†, Steven J. Orcutt

†¶ and Sriram Krishnaswamy

†‡§

†Research Institute, Children’s Hospital of Philadelphia &

‡Department of Pediatrics, University of

Pennsylvania, Philadelphia, PA 19104

Running Head: Membrane Binding and Zymogen Activation

§Correspondence: Sriram Krishnaswamy, Children’s Hospital of Philadelphia, 310 Abramson, 3615 Civic

Center Boulevard, Philadelphia, PA 19104 Voice: (215) 590-3346 Email:[email protected]

Background: Prothrombin variants lacking

membrane binding have probed the contribution

of the substrate-membrane interaction in

thrombin formation by prothrombinase.

Results: Loss of membrane binding yields

modest changes in rate but affects the pathway

for substrate cleavage.

Conclusions: Membrane binding by the

substrate constrains the presentation of

prothrombin for cleavage by prothrombinase.

Significance: New insights into how the action

of prothrombinase on prothrombin is regulated.

SUMMARY

Long-standing dogma proposes a profound

contribution of membrane binding by

prothrombin in determining the rate at which

it is converted to thrombin by

prothrombinase. We have examined the

action of prothrombinase on full-length

prothrombin variants lacking -

carboxyglutamate modifications (desGla)

with impaired membrane binding. We show

an unexpectedly modest decrease in the rate

of thrombin formation for desGla

prothrombin but with a major effect on the

pathway for substrate cleavage. Using desGla

prothrombin variants in which the individual

cleavage sites have been singly rendered

uncleavable, we find that loss of membrane

binding and other Gla-dependent functions in

the substrate leads to a decrease in the rate of

cleavage at Arg320

and a surprising increase

in the rate of cleavage at Arg271

. These

compensating effects arise from a loss in the

membrane component of exosite-dependent

tethering of substrate to prothrombinase and

a relaxation in the constrained presentation

of the individual cleavage sites for active site

docking and catalysis. Loss of constraint is

evident as a switch in the pathway for

prothrombin cleavage and the intermemdiate

produced but without the expected profound

decrease in rate. Extension of these findings

to the action of prothrombinase assembled on

platelets and endothelial cells on fully

carboxylated prothrombin reveal new

mechanistic insights into function on

physiological membranes. Cell-dependent

enzyme function is likely governed by a

differential ability to support prothrombin

binding and the variable accumulation of

intermediates from the two possible pathways

of prothrombin activation.

Thrombin, the pivotal proteinase of blood

coagulation, is produced by the proteolytic

activation of prothrombin (1). Prothrombinase,

the physiological catalyst for this reaction, is an

enzyme complex assembled by reversible

protein-protein and protein-membrane

interactions between the proteinase, factor Xa,

its cofactor, factor Va and membranes

containing phosphatidylserine (2). Membrane-

binding by the cofactor and proteinase, mediated

by specific domains, greatly enhances both the

kinetics and thermodynamics of prothrombinase

assembly (1,2). Reactions between membrane-

bound Xa and Va proceed with very high rate

constants because of approximation arising from

dimensional and orientational restrictions (3).

Tight binding interactions, further enhanced by

linkage effects allow the membrane-bound

proteins to bind each other at sub-nanomolar

concentrations (4). Prothrombin also binds

reversibly to these membranes in a Ca2+

-

http://www.jbc.org/cgi/doi/10.1074/jbc.M113.502005The latest version is at JBC Papers in Press. Published on August 12, 2013 as Manuscript M113.502005

Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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dependent fashion by virtue of -

carboxyglutamate residues at its N-terminus (5).

Consequently, the reversible interaction between

substrate and membranes is also expected to

result in its accelerated and oriented delivery to

membrane-bound prothrombinase (1,6). These

features are considered necessary for normal

hemostasis, allowing accelerated thrombin

formation on activated platelets or other cells

expressing phosphatidylserine on their outer

leaflet and localized at the site of vascular

damage (7).

The essential role for membrane binding in

blood coagulation is obvious from bleeding

associated with deficiencies in the vitamin K-

dependent reactions required for the post-

translational carboxylation of specific

glutamates to form -carboxyglutamate (8).

Warfarin and its derivatives, which interfere

with -carboxylation, are widely used for the

therapeutic control of thrombosis and need

careful dosing to avoid bleeding (9). However,

interference with -carboxylation impacts Ca2+

-

and membrane binding of all the vitamin K-

dependent coagulation proteins (9). In the case

of prothrombinase, this would impair membrane

binding by Xa as well as prothrombin, thereby

impacting the assembly of prothrombinase as

well as membrane-dependent substrate delivery.

Thus, warfarin effects do not permit incisive

inferences regarding the importance of the

substrate-membrane interaction in function.

The contribution of membrane-binding by

prothrombin towards thrombin formation has

been extensively investigated (6). Although

some controversies linger, it is widely accepted

that membrane binding by prothrombin plays an

essential role in affecting the rate of thrombin

formation. Ideas in the field are dominated by an

influential model proposed almost 30 years ago

based on co-concentration effects of

prothrombinase and prothrombin in a

microscopic sub-space bordering the membrane

surface (10,11). The model predicts a

catastrophic decrease in rate, at least by a factor

of 3,500, associated with a loss in membrane

binding by the substrate (10). The proposed

magnitude of functional loss is not fully

consistent with kinetic studies done in solution

or with various prothrombin fragments lacking

the membrane binding domain (12,13).

However, some of those interpretations could be

compromised by unanticipated effects associated

with proteolytic elimination of approximately

30% of the polypeptide structure of

prothrombin.

A complexity, insufficiently considered in the

foregoing, arises from the fact that the

conversion of prothrombin to thrombin requires

the action of prothrombinase on two sites in two

sequential enzyme catalyzed reactions (14). For

prothrombinase assembled on phospholipid

vesicles containing an optimal fraction of

phosphatidylserine, the reaction exclusively

proceeds by initial cleavage at Arg320

followed

by cleavage at Arg271

yielding meizothrombin

(mIIa)1 as an intermediate (for clarification, see

Scheme II below) (15,16). The basis for such

ordered cleavage, despite the fact that both sites

are accessible for proteolysis, lies in the

constrained way the substrate is tethered to the

enzyme through exosite interactions (17,18).

These interactions, between enzymic sites

removed from the active site and sites on the

substrate distant from the cleavage site, facilitate

the constrained presentation of the cleavage sites

for docking at the active site of Xa within

prothrombinase (14,18,19). While such ideas

have centered on protein-protein contacts, the

substrate-membrane interaction in the vicinity of

prothrombinase would also constitute a

component of exosite binding that might further

contribute to the constrained presentation of the

substrate to prothrombinase. This idea is

supported by previous studies hinting at an

altered order of bond cleavage of prothrombin

species isolated from the blood of warfarin-

treated cows and by studies done in the absence

of membranes (13,20).

We now use newer developments in the

understanding of substrate recognition by

prothrombinase as a framework to investigate

this problem. We employ a series of full length

recombinant prothrombin variants lacking -

carboxylation (desGla, dG) to permit an

assessment of the contribution of membrane

binding by the substrate on all four possible

cleavage reactions. Our findings yield surprising

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insights into how membranes regulate function

with bearing on prothrombinase function on

physiologic membranes that likely express

insufficient amounts of phosphatidylserine for

robust prothrombin binding.

EXPERIMENTAL PROCEDURES

Reagents—Human plasma used for protein

isolation was a generous gift of the

Plasmapheresis Unit of the Hospital of the

University of Pennsylvania. D-phenylalanyl-L-

proline-L-arginine chloromethyl ketone (FPRck,

Calbiochem), Alexa488-maleimide, (Invitrogen),

dansylarginine-N-(3-ethyl-1,5-pentanediyl)-

amide (DAPA, Haematologic Technologies) and H-D-phenylalanyl-L-pipecolyl-L-arginine-p-

nitroanilide (S2238, DiaPharma) were from the

indicated suppliers. Concentrations of stock

solutions prepared in water were determined

using = 8,270 M

-1.cm

-1 (S2238) and =

4,010 M-1

.cm-1 (DAPA). The acetothioacetyl

adduct of FPRck (ATA-FPRck) was prepared by

reacting FPRck to completion with an excess of

succinimidyl acetothioacetate (Invitrogen) and

purification as previously described (21). Small

unilamellar phospholipid vesicles (PCPS)

composed of 75% (w/w) hen egg L-α-

phosphatidylcholine and 25% (w/w) porcine

brain L-α-phosphatidylserine (Avanti) were

prepared and quality controlled as described

(12). Large unilamellar vesicles containing

97.5% (w/w) L-α-phosphatidylcholine and 2.5%

(w/w) L-α-phosphatidylserine were prepared by

extrusion and quality controlled as before (15).

Concentrations of PCPS were determined by

hydrolysis and colorimetric determination of

inorganic phosphate (22). Kinetic measurements

were conducted in 20 mM Hepes, 0.15 M NaCl,

0.1% (w/v) polyethyleneglycol (Mr=8K), 5 mM

CaCl2 pH 7.5 (Assay Buffer) at 25o

C. Protein

substrates were exchanged into Assay Buffer by

centrifugal gel-filtration before use.

Proteins—Prothrombin, factor X and factor V

were isolated from human plasma by established

procedures (23,24). Factor Xa and factor Va

were purified and quality controlled following

preparative activation of factor X by the purified

activator from Russell’s viper venom or of factor

V by thrombin as before (15,25). Thrombin and

prethrombin 2 (P2) were purified following

preparative proteolysis of prothrombin as

described (26). Recombinant tick anticoagulant

peptide (rTAP) was prepared as before (27).

Fully carboxylated wild type recombinant

prothrombin (IIWT) and its variants containing

Gln replacing Arg271

(IIQ271), Gln replacing

Arg320

(IIQ320) and Gln replacing Arg271

and

Arg320

(IIQQ) were produced in stably

transformed HEK293 cell lines and purified as

before (15). Uncarboxylated prothrombin

variants (dG-IIWT, dG-IIQ271, dG-IIQ320 and dG-

IIQQ) were produced by culturing the same stable

cell lines in serum free media without vitamin K.

Purification of these forms employed the scheme

utilized for the carboxylated forms except that

the pool from the first chromatography step was

precipitated with 80% saturated (NH4)2SO4

instead of barium citrate and their elution

positions in the subsequent chromatography

steps were clearly different from the

carboxylated counterparts. All recombinant

prothrombin variants were quality controlled by

N-terminal sequencing, time-of-flight mass

spectrometry and quantitative analysis of -

carboxyglutamate following base hydrolysis

(28). Uncarboxylated F12 (dG-F12) was

obtained by preparative activation of dG-IIS195A

by prothrombinase and purified using

procedures similar to those described for

carboxylated F12. Uncarboxylated prothrombin

(dG-IIWT, 32 µM) in 20 mM Hepes, 0.15 M

NaCl , 5 mM CaCl2 and 5 µM CoCl2 was

preparatively activated with 0.42 µM ecarin in

the presence of ATA-FPRck (0.16 mM) and the

resulting inactivated meizothrombin (dG-mIIai)

was isolated as before (15). A fraction of dG-

mIIai was reacted with an excess of Alexa488

maleimide in the presence of 0.1 M NH2OH and

the resulting singly labeled fluorescent adduct

(dG-mIIaAlexa488) was purified as described

(15,29).

The cDNA encoding Ecarin with Ser170 replaced

with Pro, a His6 extension at the COOH

terminus and flanked by HindIII and EcoR1 sites

was synthesized based on the published

sequence (30). Digestion with these enzymes

allowed for cloning into pcDNA 3.1+

(Invitrogen) and subsequent transfection of AV-

12 cells to obtain stable clones. Highest

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producing clones were identified by

measurements of prothrombin activation and

verified by western blotting using a mouse anti-

His-4 antibody (Qiagen). Ecarin was expressed

on a large scale in serum free medium

essentially following procedures described for

the prothrombin variants (15). Purification was

done by an initial capture step with Q-Sepharose

Fast Flow (Pharmacia) followed by

chromatography using Poros HQ-150 (Applied

Biosystems) using buffers and gradient

conditions described for prothrombin

purification (15). The pool containing Ecarin

activity was dialyzed into 20 mM Hepes, 10 mM

imidazole, 0.15 M NaCl, pH 7.4 and applied to a

1 ml HisTrap FF column (GE Healthcare)

charged with Ni2+

. Bound protein was eluted

with the same buffer containing 500 mM

imidazole, dialyzed into 20 mM Hepes, 0.15 M

NaCl, 5 µM CoCl2, pH 7.4 and stored frozen in

aliquots.

Protein concentrations were determined using

the following molecular weights and extinction

coefficients ( ): Xa, 45,300, 1.16 (31); all

prothrombin variants, 72,000, 1.47 (32);

thrombin or P2, 37,500, 1.89 (26); Va, 175,000,

1.78 (33); F12, 34,800, 1.2 (32); rTAP, 6,980,

2.56 (27); ecarin, 88,000, 1.0.

Cleavage of Prothrombin Variants—Reaction

mixtures (800 µl) containing either 5 µM or 1.4

µM prothrombin variant, 30 µM DAPA, 30 µM

PCPS, 30 nM Va in Assay Buffer at 25o

C were

initiated by the addition of either 0.4 nM or 0.15

nM Xa. Aliquots (40 µl) withdrawn at the

indicated times were quenched by mixing with

16 µl of 0.2 M Tris, 6.4% (w/v) SDS, 32% (v/v)

glycerol, 0.04% (w/v) bromphenol blue, 50 mM

EDTA, 50 mM dithiothreitol, pH 6.8

supplemented with 36 µM FPRck. Following

heating at 85 °C for 5 min., samples were

subject to electrophoresis using 10% Tris-

Glycine gels (Invitrogen), and protein bands

were visualized by staining with Coomassie

Brilliant Blue R250 for experiments done at the

high substrate concentration or by the Colloidal

Blue stain and infra-red imaging for the low

substrate concentration (34). Quenched samples

from the low concentration data set were also

visualized following quantitative western

blotting as previously described (34). For

measurements of proteolytic activity, aliquots

(10 µl) were quenched by mixing with 40 µl of

Assay Buffer containing 1 µM rTAP and 50 mM

EDTA in place of CaCl2. The concentration of

proteinase formed was determined from initial

velocities of S2238 hydrolysis as described (35).

Kinetic Studies— Initial velocity measurements

of proteinase formation from dG-IIQ271 were

determined discontinuously as previously

described (15). Reaction mixtures (200 µl)

containing the indicated concentrations of dG-

IIQ271, 30 µM PCPS and 30 nM Va at 25 °C,

were initiated with 0.5 nM Xa. Aliquots (10 µl)

were removed at 0, 0.5, 1.0, 1.5, 2 and 3 min

following initiation and quenched by mixing

with 40 µl Assay Buffer containing 1 µM rTAP

and 50 mM EDTA in place of CaCl2. The

concentration of proteinase formed in the

quenched samples was inferred from the rate of

S2238 hydrolysis and its linear dependence on

known concentrations of thrombin established

with each experiment. Initial velocities of

proteinase formation were then determined from

the linear appearance of proteinase with time

established with at least 4 of the 6 quenched

samples. For experiments with alternate

substrates or inhibitors, initial velocities with

increasing concentrations of dG-IIQ271 were

determined in the presence of the indicated fixed

concentrations of dG-IIQQ or dG-IIQ320.

Initial velocities for the conversion of the dG-

F12/P2 mixture to thrombin (cleavage at Arg320’

)

were determined in the same way with varying

concentrations of P2 and the dG-F12

concentration maintained at either 1.2 or 1.5

molar equivalents of P2.

The kinetics of action of prothrombinase on dG-

mIIai (cleavage at Arg271’

) was inferred from the

fluorescence increase seen upon conversion of

dG-mIIaAlexa488 to thrombin analogous to the

change seen in fluorescein modified but fully

carboxylated mIIa (15). Reaction mixtures (200

µl), prepared in black 96-well plates (Corning),

contained 0.1 µM dG-mIIaAlexa488 and increasing

concentrations of dG-mIIai to achieve the

indicated total concentration of substrate, 30 µM

PCPS and 30 nM Va in Assay Buffer. Reactions

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were initiated by the addition of 1 nM Xa and

product formation was monitored at 25o

C in a

Spectramax Gemini (Molecular Devices) using

EX=488 nm and monitoring broadband

fluorescence with a 510 nm long pass filter in

the emission beam. Initial, steady state velocities

of the fluorescence increase were converted to

concentration terms using the limits of the

progress curves as described (15,29).

Platelets— Platelets were isolated from blood

freshly drawn by venipuncture from aspirin-free,

volunteer donors following written consent

using a protocol approved by the institutional

review board. Blood (45 ml) was drawn into 5

ml of anticoagulant composed of 65 mM citric

acid, 85 mM Na3citrate, 2% (w/w) dextrose.

Platelet rich plasma was obtained by

conservative aspiration of part of the upper layer

after centrifugation (1000xg, 18 min, 25 o

C) in a

swinging bucket rotor. Aliquots of platelet rich

plasma (5 ml) were applied in parallel to

columns of sepharose 2B-CL300 (2.5x10.5 cm,

50 ml) equilibrated in 3.8 mM HEPES, 0.38 mM

Na2HPO4, 137 mM NaCl, 2.68 mM KCl, 0.98

mM MgCl2, 5.55 mM dextrose, 0.2% (w/v) BSA

pH 7.3 (Hepes/Tyrodes/BSA). Platelets eluting

in the void volume, well separated from the

plasma fraction, were pooled and counted using

a Hemavet 1500FS (CDC Technologies). Yields

were typically ~300,000 platelets/µl blood and

were essentially free of other cell types. Platelets

were maintained at room temperature and used

in experiments within ~2 hr of isolation.

Endothelial Cells — Human umbilical vein

endothelial cells (HUVECs, 1-7 passages) were

a generous gift of Dr. Long Zheng, Children’s

Hospital of Philadelphia. The cells were grown

to near-confluence in 6 well plates using EBM-2

medium (Lonza). Prior to the experiment,

HUVECs were washed 3 times with 3 ml of

HEPES/Tyrodes/BSA supplemented with 5 mM

CaCl2. Following washing, 0.5 ml of the same

buffer was added to the wells in preparation for

thrombin activation.

Prothrombin Cleavage on Cell Membranes—

Freshly purified platelets, adjusted to 2x108/ml,

were supplemented with 1 M stocks of Tris pH

7.5 and CaCl2 to achieve final concentrations of

20 mM and 5 mM respectively. Platelets were

activated by thrombin (10 nM, 3 min) followed

by the addition of 12 nM hirudin. Reaction

mixtures (800 µl, 25 °C) were prepared by

mixing equal volumes of the activated platelet

preparation and a solution prepared in Assay

Buffer containing 2.8 µM prothrombin variant,

60 µM DAPA and 60 nM Va. Cleavage

reactions were initiated by the addition of 0.5

nM Xa, sub-sampled at the indicated times for

SDS-PAGE analysis as above and analyzed by

quantitative western blotting (34). Washed

HUVECs were activated with thrombin (20 nM,

3 min) followed by the addition of 25 nM

hirudin. Reaction mixtures (1 ml) were prepared

in the wells of the 6-well plate by the addition of

2.8 µM prothrombin variant, 60 µM DAPA and

60 nM Va in Assay Buffer (0.5 ml). The

activation reaction was initiated by the addition

of 0.2 nM Xa and allowed to proceed with rotary

shaking (400 rpm, Thermomixer R, Eppendorf )

at 25 °C. Reaction mixtures were sampled at the

indicated times for SDS-PAGE analysis as

above and analyzed by quantitative western

blotting (34).

Data Analysis— Concentrations of PCPS and

Va were chosen to saturate Xa based on the

measured equilibrium constants for

prothrombinase assembly (4). The concentration

of enzyme was considered equal to the limiting

concentration of Xa in each experiment and used

to normalize initial velocities.

Quantitative densitometry of stained gels or

from quantitative western blotting was

performed as previously validated and described

in detail (12,15,16,34). Estimates of initial

velocity from progress curves constructed in this

way were obtained by analysis according to the

logarithmic approximation (36). Observed

steady state kinetic constants were determined

by non-linear least squares analysis according to

the Henri-Michaelis-Menten equation. Global

analysis according to Scheme I to obtain

intrinsic constants was done with the rapid

equilibrium assumption using Dynafit (Biokin)

(37). Provided this assumption holds, the

kinetics for cleavage at the individual sites in

dG-IIQ271 and dG-IIQ320 can be described by the

Henri-Michaelis-Menten equation (18,35).

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Intrinsic constants for either of the two cleavage

events are related to the observed kinetic

constant by:

(1)

( ⁄ )

(2)

where KEXO is the cleavage site independent

equilibrium dissociation constant for exosite

binding. Kmobs, (V/E)obs, Ks* and kcat would be

subscripted with either 271 or 320 depending on

the single cleavage site substrate variant used.

These relationships were used to compute terms

otherwise inaccessible from global analysis

according to Scheme I. All fitted constants are

listed ± 95% confidence limits. Errors from

fitted constants were propagated for the

calculated terms (38). With the exception of the

data set obtained for the cleavage of dG-IIQ271 or

dG-IIQ320 by Xa partially saturated by µM

concentrations of Va in solution, the reported

data are representative of two or more

experiments performed at a comparable level of

detail, frequently with different protein

preparations.

RESULTS

Prothrombin Variants— As in previous work,

we have employed recombinant variants of

prothrombin in which the individual cleavage

sites at Arg271

and Arg320

have been rendered

uncleavable, either singly or in combination, by

substitution with Gln (15). Culture of the stable

cell lines expressing these variants in the

absence of vitamin K yielded fully

uncarboxylated protein. Quantiative analysis of

-carboxyglutamic acid (Gla) content following

base hydrolysis yielded the expected 10 moles

Gla/mole protein for the proteins expressed in

the presence of vitamin K (15). In contrast, a Gla

peak was undetectable for the dG variants

providing an upper limit estimate of 0.3

Gla/mole protein. N-terminal sequencing

verified correct processing of the propeptide

during secretion and mass spectrometry yielded

the expected molecular weight for full-length

prothrombin (not shown). A desGla variant

(dG-IIWT) produced minimal changes in light

scattering when titrated with increasing

concentrations of PCPS. In a parallel

experiment, the fully carboxylated protein (IIWT)

produced a large and saturable change in light

scattering intensity signifying binding (not

shown). In accordance with the literature (20),

we estimate that the desGla variants of

prothrombin bind to PCPS membranes with at

least 500-fold weaker affinity than the fully

carboxylated protein.

Activation of Prothrombin— The contribution

of prothrombin -carboxylation to its function as

a substrate for prothrombinase was first assessed

by progress curves of thrombin formation (Fig.

1). As expected, prothrombinase yielded

thrombin at a lower rate from dG-IIWT in

comparison to IIWT (Fig. 1). However, in

contrast to the profound reduction in rate

expected based on the literature, we were

surprised to find that the initial rate of thrombin

formation from dG-IIWT was only modestly

lower by a factor of ~5 (10).

Progress curves were further analyzed by SDS-

PAGE and protein staining (Figs. 2A & 2B).

The cleavage patterns observed for IIWT (Fig.

2A) were consistent with sequential cleavage at

Arg320

to yield mIIa followed by cleavage at

Arg271

to produce thrombin as previously

reported (15). The signature features of this

cleavage pathway are the transient accumulation

of the F12-A fragment, the delayed appearance

of F12 following the conversion of mIIa to

thrombin and the lack of any detectable

prethrombin 2 (P2). In contrast, consumption of

dG-IIWT, only modestly slower than that of IIWT,

yielded no obvious evidence for F12-A

formation but instead produced P2 in a transient

way (Fig. 2B). Thus, the modestly decreased

rate of thrombin production from dG-IIWT (Fig.

1) obscures major differences in the way the

substrate is cleaved by prothrombinase.

Nevertheless, proteinase formation as judged by

peptidyl substrate cleavage from either

carboxylated or uncarboxylated prothrombin

matches well with the appearance of the

thrombin B chain determined by quantitative

densitometry (Fig. 1).

Cleavage of the Individual Bonds within

Prothrombin— Initial evidence implying altered

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selectivity for bond cleavage within dG-IIWT by

prothrombinase was further pursued by studies

with carboxylated and uncarboxylated versions

of the cleavage site mutants (Fig. 2 C-F). IIQ271

was rapidly consumed by prothrombinase from

cleavage at Arg320

to produce mIIa (Fig. 2C).

The use of dG-IIQ271 yielded much slower

cleavage at Arg320

(Fig. 2D). Cleavage at Arg271

in IIQ320 proceeded very slowly (Fig. 2E)

reflecting the preference of prothrombinase for

cleavage at Arg320

in intact prothrombin (15).

We were surprised to find that dG-IIQ320 was

cleaved at a greater rate than IIQ320 (Fig. 2F).

While this observation has bearing on the altered

cleavage patterns seen with dG-IIWT, it also

illustrates that loss of membrane binding by the

substrate is not uniformly deleterious and

unexpectedly produces a gain in a selected

aspect of function.

Because F12 from the desGla variants migrates

as an indistinct smear, initial studies were

conducted at 5 µM substrate to facilitate reliable

interpretation of SDS-PAGE analyses (Fig. 2).

To rule out the possibility that our unexpected

findings may reflect a peculiarity of this choice

in concentration, we pursued further work at the

physiological concentration of prothrombin (1.4

µM) using infra-red detection of stained gels or

by quantitative western blotting with near-IR

fluorescence detection (34). The findings were

equivalent, borne out by the quantitative analysis

of prothrombin consumption from the two types

of measurements (Fig. 3). Loss of membrane

binding in IIWT only yielded a modest ~2.5 fold

decrease in initial rate of prothrombin

consumption (Fig. 3A). This modest decrease

arose from opposing effects producing a larger

decrease in rate (~15-fold) for cleavage at Arg320

in dG-IIQ271 (Fig. 3B) but an increase in rate (~4-

fold) for cleavage at Arg271

in dG-IIQ320 (Fig.

3C).

Initial rates of prothrombin consumption (Table

1) illustrate that prothrombinase consumes

carboxylated prothrombin by preferential

cleavage at Arg320

with a minor contribution

from cleavage at the alternate site. In contrast,

prothrombinase acts on dG-IIWT at a slightly

reduced rate but by preferential cleavage at

Arg271

while cleavage at Arg320

also proceeds at

a significant rate. This carboxylation-dependent

switch in selectivity, thus far interpreted to

reflect the contribution of membrane binding by

prothrombin, accounts for the change in

cleavage patterns seen in the action of

prothrombinase on IIWT and dG-IIWT (Fig. 2 A &

B).

Altered Cleavage Site Selectivity in desGla

Prothrombin— Further kinetic analyses using

the initial velocity and rapid equilibrium

assumptions were pursued using a model

developed in previous studies with carboxylated

prothrombin (Scheme I). This model is rooted in

kinetic and equilibrium binding measurements

illustrating that equivalent exosite binding

interactions are responsible for tethering the

substrate to the enzyme regardless of cleavage

site (16,39). Active site docking by Arg271

in

exosite-bound IIQ320 or Arg320

in enzyme-bound

IIQ271 then occurs in a mutually exclusive way

for catalysis at the individual sites (Scheme I).

IIQQ is uncleavable because it cannot engage the

active site of prothrombinase (39). Alternate

substrate studies measured the rate of dG-mIIa

formation from varying concentrations of dG-

IIQ271 in the presence of different fixed

concentrations of dG-IIQ320 or dG-IIQQ (Fig. 4).

Global analysis of the data according to Scheme

I yielded adequate fits and permitted meaningful

assessment of KEXO, Ks*320, Ks*271 and kcat320

(Fig. 4). Because the product of dG-IIQ320

cleavage is not measured here, kcat271 was

estimated in combination with initial rates

determined from densitometry measurements

(Table 1). These parameters reflect the intrinsic

constants governing the ability of

prothrombinase to discriminate between the two

sites within intact but desGla prothrombin

(Table 2).

Comparisons with values previously determined

for fully carboxylated prothrombin (Table 2),

reveal how impaired membrane binding by

desGla prothrombin affects its recognition by

prothrombinase. For the desGla variants, exosite

binding is ~70-fold weaker than for the

carboxylated substrate. Membrane binding and

possibly other Gla-related functions play a major

role in the exosite-dependent tethering of

prothrombin to prothrombinase. In fully

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carboxylated prothrombin, the unimolecular

binding constant for active site docking by

Arg320

(Ks*320) is ~200-fold more favorable than

for Arg271

(Ks*271) (Table 2). Such

discrimination is lost in desGla prothrombin

wherein active site docking by Arg320

and Arg271

occurs with approximately equal and low

affinity (Ks*320 Ks*271 2) (Table 2). It would

appear that in desGla prothrombin, the

constrained presentation of the substrate for

active site docking has been altered, resulting in

the scrambling of preferential active site docking

of one cleavage site over the other. The intrinsic

kcat for cleavage at Arg320

remained unaffected

by the carboxylation state of the substrate.

Surprisingly, kcat271 was increased ~30-fold for

the desGla variant in comparison to

carboxylated prothrombin (Table 2). This

implies that loss of membrane binding and/or

other Gla-related functions mediated by the N-

terminus of prothrombin detectably affect distant

structures surrounding the Arg271

site.

Kinetics of the Four Possible Cleavage

Reactions— The observed steady state kinetic

constants for each of the four possible half-

reactions allows prediction of flux towards

thrombin formation. Steady state kinetic

constants were measured and/or calculated for

the action of prothrombinase at Arg271

and

Arg320

in otherwise uncleaved desGla

prothrombin (Table 2). Initial velocity studies

were also performed to examine the action of

prothrombinase at the remaining site in singly

cleaved desGla variants (Fig. 5). Steady state

kinetic constants for the cleavage at Arg271’

following initial cleavage at Arg320

were

obtained using dG-mIIa as substrate (Fig. 5A).

Cleavage at Arg320’

following initial cleavage at

Arg271

was assessed using P2 reconstituted with

dG-F12 (Fig. 5B). Equivalent initial velocities

obtained at two ratios of dG-F12:P2 support the

contention that P2 was essentially saturated with

dG-F12 at all concentrations used (Fig. 5B).

Observed steady state kinetic constants (Table

2) with representative values listed in Scheme II

provide the basis for the further consideration of

the action of prothrombinase on desGla

prothrombin.

Initial rates calculated at the physiological

concentration of prothrombin reveal that desGla

substrate forms are cleaved more slowly (by at

least a factor of 10) than the equivalent

carboxylated species for three of the four

possible half-reactions (Scheme II). The

exception is cleavage at Arg271

in intact

prothrombin that is increased for desGla

substrate in accordance with the rates detailed

for dG-IIQ320 (Table 1). Thus, asymmetry in

action of prothrombinase at Arg271

in intact

prothrombin versus mIIa in the carboxylated

forms is altered for the desGla substrate (15).

Consequently, ~80% of the rate of consumption

of desGla prothrombin is expected to result in

the formation of dG-F12/P2 and ~20% from the

formation of dG-mIIa. In accordance with

observations (Fig. 2), minor amounts of dG-

mIIa are predicted to accumulate as an

intermediate while abundant amounts of P2 are

expected as a long lasting intermediate given its

slower conversion to thrombin. Simulations with

these steady state kinetic constants (not shown)

indicate that the initial rate of proteinase

formation (mIIa+IIa) through the initial cleavage

of desGla prothrombin at Arg320

would be ~6-

fold greater than proteinase formation via the P2

pathway. This point highlights the dangers

inherent in inferring the predominant pathway

for product formation in such systems solely on

the basis of amounts of intermediate observed.

Correlation with Membrane Binding— A

diagnostic difference in the action of

prothrombinase on desGla versus fully

carboxylated substrate lies in the relative rates

for cleavage at the two sites within intact

prothrombin (Scheme II). In the case of

carboxylated substrate cleavage at Arg320

is ~30-

fold greater than cleavage at Arg271

. For desGla

prothrombin, cleavage at Arg271

is ~4-fold

greater than cleavage at Arg320

. At issue is

whether such effects can be wholly ascribed to a

loss in membrane binding by the desGla variants

rather than other -carboxyglutamate-dependent

effects on the substrate.

To address this uncertainty, we determined the

relative rates of cleavage at the two sites within

fully carboxylated prothrombin but in the

absence of membranes. Progress curves for the

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cleavage of IIQ320 and IIQ271 in the absence of

added membranes were constructed by

quantitative blotting following the addition of 1

nM Xa partially saturated (~50%) with 2 µM Va

(Fig. 6A). Comparable initial rates were

obtained for cleavage at the individual sites in

otherwise fully carboxylated intact prothrombin

(Fig. 6A). While the ~30-fold discrimination

seen for cleavage at Arg320

relative to Arg271

seen for the action of membrane bound

prothrombinase on carboxylated substrate was

abolished in the absence of membranes, this

reaction system only partly replicated the

findings seen with the desGla prothrombin

variants (Table 1). Cleavage at Arg271

in IIQ320

yielded v/E equivalent to that observed for the

action of membrane-bound prothrombinase on

dG-IIQ320. Therefore, the gain in function seen

for cleavage at Arg271

in intact desGla

prothrombin and only a fraction of the loss in

function seen for cleavage at Arg320

can be

replicated with fully carboxylated prothrombin

but in the absence of membranes. Since

essentially equivalent results were obtained

using dG-IIQ320 and dG-II271 (Fig. 6A) the data

implicate subtle differences between membrane

assembled prothrombinase and Xa saturated

with Va in solution as the reason for the

discrepancy. The findings are consistent with the

conclusion that membrane binding by the

substrate contributes in a major way to its

constrained presentation to prothrombinase and

the subsequent ability of the enzyme to

discriminate between the two cleavage sites.

Cleavage Site Selectivity on Physiological

Membranes— Relative rates of cleavage of

fully carboxylated IIQ271 and IIQ320 were

employed to investigate how the substrate-

membrane interaction may control

prothrombinase function on physiological

surfaces. This comparative approach allows

secure conclusions without knowledge of the

concentration of productively-bound

prothrombinase. Studies with activated platelets

revealed that cleavage at either Arg320

or Arg271

in intact prothrombin proceeded at equivalent

rates (Fig. 6B). This indicates that

prothrombinase assembled on the platelet

surface does not discriminate between the two

cleavage sites in prothrombin much as Xa bound

to Va in the absence of membranes (Fig. 6A).

Thus on the platelet surface, prothrombin

consumption is expected to proceed

approximately equally through the P2 and mIIa

pathways. In contrast, when assembled on

activated HUVECs, prothrombinase acted on

Arg320

in IIQ271 ~2-fold faster than it cleaved

Arg271

in IIQ320 (Fig. 6C). For these cells,

cleavage of prothrombin would partition

between the formation of mIIa and P2 in a ratio

of 2:1. The findings are consistent with a greater

contribution from membrane-binding by

prothrombin on HUVECs in comparison to

platelets. The exact contribution of the two

possible pathways to thrombin formation would

require knowledge of the kinetic constants for all

four cleavage reactions. However, the

implications are that variable contributions of

prothrombin cleavage via both possible

pathways will determine the amount of

intermediates observed in a cell type-dependent

fashion. Such observations might be expected to

correlate with the variable ability of the

membrane surfaces to support prothrombin

binding.

These predictions were tested by quantitative

western blotting to analyze IIWT cleavage by

prothrombinase assembled on platelets or

HUVECs previously activated with thrombin.

With platelets, bands corresponding to both P2

and mIIa (F12-A) accumulated transiently (Fig.

7A). In agreement with prior work, only a trace

band arising from mIIa (F12-A) was evident

(Fig. 7A) (40). In contrast, bands corresponding

to mIIa and P2 were more prominent with

HUVECs yielding approximately equal peak

concentrations of F12-A and P2 (Fig. 7B). In

order to relate these findings to the limited

ability of these activated cells to support

prothrombin binding, we pursued studies with

IIWT and synthetic vesicles containing 2.5%

(w/w) phosphatidylserine. The intent was to

replicate the low amounts of phosphatidylserine

expected in the outer leaflet of these cells which

would facilitate the high affinity interactions

required for prothrombinase assembly but

significantly impact prothrombin binding and

density of bound substrate (7). Accordingly,

bands corresponding to the transient formation

of both P2 and mIIa were observed with these

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synthetic membranes approximating the pattern

of cleavage seen with the activated cells (Fig.

7C). Taken together with predominant cleavage

of IIWT via the formation of mIIa seen with

vesicles containing 25% (w/w)

phosphatidylserine (Fig. 2A), the data present a

consistent picture implicating a limited but

variable ability of physiological membranes to

support substrate binding as a major determinant

of the pathway for prothrombin activation.

DISCUSSION Our strategy of using appropriate recombinant

prothrombin derivatives to probe the details of

the newly expanded model for the action of

prothrombinase on prothrombin now sheds new

light on a long-standing problem in coagulation

enzymology. Prevailing dogma predicts a

catastrophic decrease in the rate of action of

prothrombinase on prothrombin with impaired

membrane binding. Instead, by the use of full-

length prothrombin variants lacking Gla

modifications, we find only modest changes in

rate attributable to a loss in membrane binding

by the substrate at its physiological

concentration. These modest decreases belie

major mechanistic changes in the way

prothrombin is recognized by prothrombinase.

The findings reveal unexpected insights into

how the interaction between the substrate and

membrane in the vicinity of prothrombinase is a

major determinant of the constrained

presentation of prothrombin to the membrane-

assembled enzyme complex.

Membrane binding by the substrate, lost in the

desGla variants, contributes in a prominent way

to the initial exosite-driven tethering of the

substrate to prothrombinase (Scheme I). This

point is evident from a ~70-fold increase in KEXO

for the desGla variants. High selectivity (~200-

fold) for subsequent active site docking by

Arg320

over that of Arg271

seen with carboxylated

prothrombin is lost in the desGla variants. For

these species with impaired membrane binding,

the unimolecular binding constants for active

site docking indicate that two cleavage sites can

engage the active site of Xa within

prothrombinase with approximately equal

probability. Scrambling of selectivity for the two

cleavage sites is consistent with the loss of a

subset of exosite binding interactions that

otherwise position bound substrate in a

constrained way for preferential active site

docking by Arg320

. Coupled with the increased

intrinsic kcat271 these altered constraints in

exosite tethering of the substrate provide the

mechanistic basis for the qualitative change in

cleavage pattern observed in the action of

prothrombinase on IIWT versus dG-IIWT.

We intentionally qualify inferences of the

contribution of the substrate-membrane

interaction to function from studies with the

desGla variants. Altered function of these

variants could arise from a loss of additional

Gla-dependent functions beyond simply a loss in

membrane binding. In support of this possibility

are the findings with fully carboxylated

prothrombin variants and Xa partially saturated

with Va in solution that do not fully replicate the

observations made with desGla variants and

membrane assembled prothrombinase. However,

the fact that equivalent rates of consumption

were observed with IIQ271, IIQ320, dG-IIQ271 and

dG-IIQ320 in the absence of membranes suggests

that this small discrepancy most likely lies in

differences in the properties of Xa saturated with

Va in solution relative to the membrane

assembled enzyme. A second concern is

reflected by the previously documented

interaction between a peptide derived from the

prothrombin Gla domain and factor Va (41).

Again, compromised interactions between

desGla prothrombin variants and Va are unlikely

to be a major source of the present findings

based on the equivalent rates of consumption of

the fully carboxylated and desGla variants in the

absence of membranes (Fig 7A). However, the

need for cautious interpretation is suggested by

the obvious effects of the loss of Gla on the

intrinsic kcat for cleavage at Arg271

. It remains

uncertain whether this solely arises from

alterations in the constrained way that the

exosite-tethered substrate is presented to

prothrombinase.

Qualifications notwithstanding, our findings

ranging from studies with desGla prothrombin

variants, to studies with fully carboxylated forms

in the absence of membranes, and with

prothrombinase assembled on physiological

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surfaces or on synthetic membranes with

limiting amounts of phosphatidylserine are

internally consistent. They portray a unifying

picture wherein membrane binding plays an

essential role in dictating the presentation of

substrate to prothrombinase. Altered interactions

that affect these constraints are evident as

changes in the intermediates observed and an

apparent change in the pathway for prothrombin

cleavage. These ideas highlight, both empirically

and conceptually, the dangers in interpreting the

major pathway for thrombin formation from the

relative abundance, or lack thereof, of the two

intermediate species (40,42). They also likely lie

at the heart of some of the variable results and

recent controversies in the field (43-45).

Recent studies have proposed a fundamental

difference in the molecular architecture of

prothrombinase assembled on platelets relative

to synthetic membranes based on the formation

of prethrombin 2 as an intermediate and the lack

of observable intermediates in the fluid phase

(42). An adequate kinetic explanation for these

findings as well as justification for the major

pathway for thrombin formation will need to

await the determination of steady state kinetic

constants of each of the four possible cleavage

reactions. However, our results, point to the

inefficient binding of prothrombin to activated

platelet membranes as a parsimonious

mechanistic explanation. Parallels in the

selectivity of prothrombinase for prothrombin in

the absence of membranes and the ability of low

phosphatidylserine containing vesicles to

replicate the findings of prothrombin cleavage

seen on physiological membranes lend support

to this contention. Mechanistic issues aside, the

findings with HUVECs and platelets illustrate

that different cell types may variably support the

formation of mIIa during thrombin formation

perhaps reflecting a differential ability to

support prothrombin binding (46-48). Given its

zymogen-like character and skewed substrate

specificity of mIIa for the anticoagulant

activities of thrombin, this may have bearing on

the spatial regulation of coagulation (34).

Our findings contrast with prevailing dogma

associating impaired membrane binding by

prothrombin with a profound loss in the rate of

thrombin formation (10,11). Indeed, when

considered in the context of the four possible

reactions of prothrombin activation, the desGla

substrate variants exhibit a very modest decrease

in rate for three of the four cleavage reactions.

The increased rate observed for one of the steps

even more surprisingly illustrates that loss of

Gla-dependent functions is not uniformly

deleterious to the function of prothrombin as a

substrate for prothrombinase. Clearly, bleeding

associated with incorrect dosing with warfarin

cannot be ascribed to the faulty function of

desGla prothrombin as a substrate. However, the

reduced amounts of anticoagulant-specific mIIa

produced as an intermediate during the

activation of desGla prothrombin could have

bearing on warfarin-induced thrombosis (9).

It could also be argued that prothrombin

completely devoid of Gla is not a good facsimile

of partially carboxylated zymogen forms

expected in the blood of patients being

therapeutically treated with warfarin. This

argument is weak because an average Gla

content of 3 moles/mole protein seen in the non-

membrane binding fraction of prothrombin from

treated individuals reveals nothing of the

fractional distribution of the various

prothrombin isoforms with a Gla content

varying between 0 and 10 (49). It should be

noted that prothrombin devoid of Gla has been

isolated from the blood of warfarin-treated cows

(20). In keeping with the cooperative nature of

Ca2+

binding by the Gla residues, mixtures of

bovine prothrombin isoforms lacking 3-4 of the

full Gla complement of 10 were seen to exhibit

greatly impaired membrane binding (20).

A recent structure of a variant of prothrombin

lacking residues 1-44 encompassing the Gla

domain from the Di Cera laboratory has

provided evidence for flexibility in the linker

between the fragment 1 and fragment 2 domains

as well as the disorder in the region surrounding

the Arg271

cleavage site (50). Such flexibility

may provide an explanation for how the desGla

substrate tethered to prothrombinase, in the

absence of additional constraints imposed by

membrane binding, may allow active site

docking of two distant sites with approximately

equal probability. However, the associated claim

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that Arg271

is solvent accessible while Arg320

is

buried has yielded the major conclusion that

prothrombinase must cleave first at Arg271

before the Arg320

site becomes exposed (50).

This conclusion regarding the molecular

mechanism of prothrombin activation cannot be

reconciled with a large body of evidence

regarding the cleavage of IIWT by

prothrombinase on synthetic PCPS membranes

(15,17,43,51,52). The present work further rules

out the relevance of such a conclusion for

prothrombinase function on natural membranes

(platelets or HUVECs), in solution or even on its

action on desGla-IIWT. We are nonplussed by the

obvious disparity between the structure-based

proposal and the existing literature particularly

as the authors cite a paper from 1872 yet fail to

acknowledge a broad swathe of contemporary

work in the field that runs counter to their claim

(15,17,43,51-54).

In summary, our studies with desGla

prothrombin variants provide surprising insights

into a long-standing problem in coagulation

enzymology. The findings point to a prominent

role for the substrate-membrane interaction in

mediating exosite-dependent binding to

prothrombinase and the constrained presentation

of the substrate for cleavage. Our findings bear

on how the varied ability of physiological

membranes to affect such constrained

presentation of prothrombin might underlie the

regulation of the pathway for prothrombin

cleavage and the intermediate produced.

Whether such differential proportioning via the

formation of mIIa relative to the zymogen P2

has a significant regulatory role remains to be

established.

ACKNOWLEDGEMENTS

This work was supported by grants HL-074124 and HL-108933 (to SK) from the NIH. We acknowledge

the assistance of Long Zheng in providing HUVECs and expertise with their culture. We are also grateful

to Rodney Camire and William Church for critical review of the manuscript.

FOOTNOTES ¶ Present address: Janssen Pharmaceuticals Inc., Spring House, PA

1Abbreviations:

DAPA: dansylarginine-N-(3-ethyl-1,5-pentanediyl)-amide

desGla, dG: lacking -carboxyglutamic acid

F12: fragment 1.2 (prothrombin residues 1-271)

F12-A: fragment 1.2-A chain (prothrombin residues 1-320)

FPRck: D-phenylalanyl-L-proline-L-arginine chloromethyl ketone

ATA-FPRck: acetothioacetyl-FPRck

Gla: containing -carboxyglutamic acid

HUVECs: human umbilical vein endothelial cells

IIWT: wild type and fully -carboxylated human prothrombin

IIQ271: fully -carboxylated human prothrombin containing Gln in place of Arg271

IIQ320: fully -carboxylated human prothrombin containing Gln in place of Arg320

IIQQ: fully -carboxylated human prothrombin containing Gln in place of Arg271

and Arg320

mIIa: meizothrombin (prothrombin cleaved at Arg320

with residues 1-320 and 321-579 in

disulfide linkage)

mIIai: mIIa covalently inactivated with ATA-FPRck

P2: prethrombin 2 (prothrombin residues 272-579)

PCPS: small unilamellar vesicles composed of 75% (w/w) L-α-phosphatidylcholine and 25%

(w/w) L-α-phosphatidylserine

PS: L-α-phosphatidylserine

rTAP: recombinant tick anticoagulant peptide

S2238: H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroanilide

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FIGURE LEGENDS

Figure 1. Proteinase Formation from Carboxylated and Uncarboxylated Prothrombin. Progress curves

for proteinase formation by prothrombinase were measured with 0.4 nM Xa, 30 nM Va, 50 µM PCPS and

5.0 µM IIWT () or dG-IIWT (). Product formation was measured by initial velocities of S2238

hydrolysis (closed symbols) or by the amount of B-chain determined by quantitative densitometry

following SDS-PAGE (open symbols). Product concentration was normalized to the initial concentration

of substrate and the lines are arbitrarily drawn.

Figure 2. Cleavage of Carboxylated and Uncarboxylated Prothrombin Variants. Samples from reaction

mixtures containing 0.4 nM Xa, 30 nM Va, 30 µM DAPA, 50 µM PCPS and 5.0 µM indicated

prothrombin variant were analyzed by SDS-PAGE following disulfide bond reduction and visualized by

staining with Coomasie Blue R250. Reaction times (in minutes) following initiation with Xa are listed on

the bottom margin. Relevant prothrombin fragments are identified on the left margin and molecular

weights of standards (x103) are shown on the right margin. Images of two gels have been aligned in each

panel.

Figure 3. Progress Curves for Prothrombin Consumption. Samples obtained from reaction mixtures

containing 0.15 nM Xa, 30 µM PCPS, 30 nM Va, 30 µM DAPA and 1.4 µM prothrombin variant were

analyzed by SDS-PAGE. The fate of the indicated prothrombin variant was inferred from densitometry

analysis of gels with colloidal blue (open symbols) or western blotting (closed symbols). Each panel

illustrates the fate of the indicated fully carboxylated () or desGla () variant normalized to the

starting concentration. The lines were arbitrarily drawn. Initial rates of prothrombin consumption are

listed in Table 1.

Scheme I. The Action of Prothrombinase on Intact Prothrombin. The substrate is initially tethered to

prothrombinase (E) by exosite binding (KEXO) independent of cleavage site availability. Arg320

in IIQ271

then engages the active site in a unimolecular binding step (Ks*320=E.IIQ271/E*.IIQ271) followed by

catalysis (kcat320) to yield mIIa. Equivalent steps determine active site docking (Ks*271= E.IIQ320/E*.IIQ320)

and catalysis (kcat271) by Arg271

in IIQ320. The double mutant (IIQQ) participates in exosite binding but does

not engage the active site of prothrombinase.

Figure 4. Kinetics of the Action of Prothrombinase on dG-IIQ271. Initial velocities of proteinase

formation were determined in reaction mixtures containing increasing concentrations of dG-IIQ271, 30 µM

PCPS, 30 nM Va, 0.5 nM Xa and different fixed concentrations of dG-IIQ320 (Panel A) or dG-IIQQ (Panel

B). Fixed concentrations of dG-IIQ320 correspond to 0, 5, 15, 20 and 30 µM (top to bottom, Panel A) and

of dG-IIQQ correspond to 0, 10, 20 and 40 µM (top to bottom, Panel B). The lines are drawn following

global analysis according to Scheme I with fitted constants listed in Table 2.

Figure 5. The Action of Prothrombinase on Singly Cleaved desGla Intermediates. Panel A: Initial

velocities for cleavage at Arg271’

in dG-mIIa were determined by fluorescence measurements using

reaction mixtures containing 0.1 µM dG-mIIaAlexa488, increasing concentrations of dG-mIIai, 30 µM PCPS,

30 nM Va and 1 nM Xa. Panel B: Initial velocities of thrombin formation measured using increasing

concentrations of P2 premixed with either 1.2 () or 1.5 () equivalents of dG-F12, 30 µM PCPS, 30

nM Va and 0.5 nM Xa. The lines are drawn following analysis using the observed steady state kinetic

constants reported in Table 2.

Scheme II. Kinetic Constants for the Four Half-Reactions of Prothrombin Activation. Observed steady

state kinetic constants for the action of prothrombinase (E) on desGla substrate species are listed for each

half-reaction. Relative rates for the carboxylated (Gla) versus uncarboxylated (desGla) substrate forms are

those calculated at 1.4 µM substrate.

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Figure 6. Membrane-Dependent Discrimination Between Arg320

and Arg271

by Prothrombinase. Progress curves for the consumption of 1.4 µM IIQ271 by cleavage at Arg

320 () or of 1.4 µM IIQ320 by

cleavage at Arg271

() were determined by quantitative western blotting using reaction mixtures

containing 1 nM Xa and 2 µM Va in the absence of membranes (Panel A), 0.5 nM Xa, 30 nM Va and 108

thrombin activated platelets/ml (Panel B) or 0.2 nM Xa, 30 nM Va and confluent HUVECs activated by

thrombin (Panel C). Panel A also illustrates data obtained with 1.4 µM dG-IIQ271 () or dG-IIQ320 ()

under the same conditions as used for the fully carboxylated variants. The lines are arbitrarily drawn.

Figure 7. Cleavage of Prothrombin by Prothrombinase assembled on Physiological Membranes. Western blots imaged with near-IR fluorescence illustrate the fate of 1.4 µM IIWT following cleavage

using Panel A: 0.5 nM Xa, 30 nM Va and 108 thrombin activated platelets/ml; Panel B: 0.2 nM Xa, 30

nM Va and confluent HUVECs activated by thrombin and Panel C: 0.5 nM Xa, 30 nM Va and 30 µM

extruded membranes containing 2.5% phosphatidylserine and 97.5% phosphatidylcholine. Reaction times

(in minutes) are listed in the lower portion of each panel. Two images have been aligned in each panel.

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Table 1: Normalized Rates of Prothrombin Consumption by Prothrombinasea

Prothrombin Derivative Bond(s) Cleaved v/E (1.4 µM substrate) v/E (5 µM substrate)

IIWT Arg320, Arg271 0.99 1.00 IIQ271 Arg320 1.14 1.09 IIQ320 Arg271 0.07 0.04 dG-IIWT Arg320, Arg271 0.39 0.63 dG-IIQ271 Arg320 0.08 0.15 dG-IIQ320 Arg271 0.29 0.57

a Initial rates of prothrombin consumption were measured from progress curves determined by quantitative densitometry following SDS-PAGE at either 1.4 µM or 5 µM substrate. Initial rates were normalized for differences in the concentration of enzyme (v/E) and are expressed in multiples of the rate observed at 5 µM IIWT.

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Table 2: Kinetic Constants for the Cleavage of Carboxylated and Uncarboxylated Substrate

Substrate Constant Units desGla Substrate

Source Gla Substrate

Source

Intrinsic Constants

KEXO µM 17.2 ± 0.5 1 0.25 ± 0.01 7 IIQ271 Ks*320 Dimensionless 1.9 ± 0.2 1 0.02 ± 0.002 8 IIQ271 kcat320 s-1 109 ± 7 1 94 ± 2 8 IIQ320 Ks*271 Dimensionless 1.7 ± 0.2 1 4.3 ± 0.11 8 IIQ320 kcat271 s-1 375 ± 78 2 ~13 9

Observed Constants

IIQ271 Kmobs,320 µM 11.2 ± 1.2 3 0.18 ± 0.01 10

11.0 ± 0.8 4 (V/E)320 s-1 38.3 ± 0.6 3 94 ± 2 10

37.4 ± 0.2 4 IIQ320 Kmobs,271 µM 10.8 ± 1.6 3 0.35 ± 0.01 10 (V/E)271 s-1 139 ± 13 5 ~ 3 10 F12/P2 Kmobs,320’ µM 20.1 ± 0.7 6 0.18 ± 0.01 10 (V/E)320’ s-1 13.5 ± 0.3 6 76 ± 1 10 mIIai Kmobs,271’ µM 11.3 ± 0.9 6 0.28 ± 0.02 10 (V/E)271’ s-1 67.0 ± 2.9 6 114 ± 3 10

1. Globally fitted according to Scheme 1 and Figure 4. 2. Calculated from (V/E)271 and Equation 2. 3. Calculated using globally fitted parameters and Equations 1 and 2. 4. Steady State constants measured in the absence of inhibitor (Fig. 4A). 5. Calculated from rates in Table I, Kmobs,271, Kmobs,320 and (V/E)320. 6. Measured in Figs 5A and 5B. 7. Representative Ki for inhibition by IIQQ, taken from reference 15. 8. Derived from direct binding measurements taken from reference 16. 9. Estimated from (V/E)271 (taken from reference 15) and Equation 2. 10. Taken from reference 15.

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Figure 1

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Figure 2

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Figure 3

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Scheme I

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Figure 4

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Figure 5

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Scheme II

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Figure 6

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Figure 7

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Harlan N. Bradford, Steven J. Orcutt and Sriram KrishnaswamyProthrombinase For Cleavage.

Membrane Binding by Prothrombin Mediates its Constrained Presentation to

published online August 12, 2013J. Biol. Chem.

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membrane binding by prothrombin mediates its constrained

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