Urry D.W. (Ed.) What Sustains Life? : Consilient Mechanisms for Protein-Based Machines and Materials

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7.4 Blood Clotting: Poised at the Cusp of Insolubility

285

c-c

Central Domain

si/

FIGURE

7.27. (A) Schematic representation of the

structure of one molecule of fibrinogen, showing

disulfide bridges and N-end-to-N-end arrangement

of chains. (Adapted with permission from Voet and

Voet.^^^) (B) Crystal structure of one side of the

native chicken fibrinogen molecule with gray, light

gray, and white for dehneation of the a-, (3-, and y-

chains, respectively, where p- and y-chains are shown

to have globular components. Soft fibrin clot forms

on removal of a 16 residue A peptide at the central

domain exposing a new amino-terminal a-chain

GPRP sequence that initiates association at the

hydrophobic y-globule EA site (shown in stereo in

Figure 7.32A), and on removal of a 14 residue B

peptide at the central domain to expose the p-chain

GHRP sequence for association with the hydropho-

bic P-globule EB site (shown in stereo in Figure

7.32B). (C) Crystal structure of the same side of

the native chicken fibrinogen molecule for the

purpose of delineating hydrophobicity with the most

hydrophobic (nonhistidine) aromatic residues in

black, other hydrophobic residues in gray, neutral

residues in fight gray, and the charged residues, acidic

and basic (including histidine), as white. It is obvious

even at this gross level of examination that the car-

boxyl-terminal globular components of the p- and

y-chains are much more hydrophobic than the

remainder of the molecule. (B and C prepared using

the crystaUographic results of Yang et al.^^ as

obtained from the Protein Data Bank, Structure File

IMIJ.)

286

Biology Thrives Near a Movable Cusp of Insolubility

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7.4 Blood Clotting: Poised at the Cusp of Insolubility

end-to-end DD docking site

287

^^E^site

Eg site for CHRP

of p-N-terminus

Central Domain

tir a'-N-terminus

site for CHRP

rf P-N-terminus

end-to-end DD docking site

^<-E^

site for GPRP

of a-N-terminus

FIGURE 7.28. Stereo view of fibrinogen, (aPY)2,

showing three sites for intermolecular contacts

resulting in formation of the fibrin clot: (1) the end-

to-end docking site for linear fiber growth that works

in cooperation with association at the

site; (2) the

site of the carboxyl-terminal y-globule hydropho-

bic domain, which provides for fibrin formation in a

second dimension due to association with the GPRP

of an a-amino terminus (at the central domain) that

was exposed on removal of the

residue A peptide;

and (3) the

site of the carboxyl-terminal P-globule

hydrophobic domain, which provides for fibrin

formation in the third dimension by association with

GHRP of

p-amino terminus (at the central domain)

that becomes exposed on removal of the 14 residue

peptide.

These,

as well as the end-to-end polymer-

ization, are all sites of hydrophobic association as

shown in Figures 7.31 and 7.32. (Prepared using the

crystallographic results of Yang et al.^^ as obtained

from the Protein Data Bank, Structure File IMIJ.)

indicated as the central domain. Front and back

sides of the fibrinogen molecule are shown in

Figure

7.27B

as obtained from a crystal struc-

ture that is 65% water. Interestingly, the phase-

separated state of poly(GVGVP) is 63% water

by weight.^^ Of the 1,364 amino acid residues of

the half molecule of chicken fibrinogen, 272

residues of the carboxyl-terminal sequence of

the a-chain are missing; at the amino termini of

the three chains 96 residues are missing, and 16

288

Biology Thrives Near a Movable Cusp of Insolubility

residues are not seen at the carboxyl terminus

of the y-chain. These missing sequences are

so disordered as to provide no diffraction

pattern.

The absence of the residues noted does not

appear to block an understanding of the

key elements of fibrin formation as much as

might have been initially expected. Binding of

the key residue sequences that become exposed

on removal of the A and B peptides identifies

the

and

sites for intermolecular associa-

tion.

The sequence GPRP, w^hich binds at the

site,

and the sequence GHRP, which binds at

the EB site, are found in the expected sites as

labeled in Figure 7.27B, w^here they look much

like black dots in this whole-molecule view.

The primary message of this consideration of

blood clotting in relation to the movable cusp

of insolubiUty becomes apparent in this whole-

molecule view of fibrinogen in Figure 7.27C.

Even at this level of inspection with the most

hydrophobic amino acid residues given in black

in Figure 7.27C, the greater hydrophobic char-

acter of the carboxyl-terminal globular units of

the y-chain and p-chains is apparent. This aspect

is pursued in more detail below when consid-

ering the double fragment D from human fibrin

itself,

which is the end-to-end association of the

y-chain globular units.

7.4.2.2

Tf-based Hydrophobicity Plots of

Human Fibrinogen Chains

The single residue Tt-based hydrophobicity

plot and the mean residue hydrophobicity plot

reported for the chains of fibrinogen provide

interesting insights. A gUmpse of the single

residue plot provides immediate sequence

insights that are easier to glean from such a plot

than from the sequences as listed in Table 7.1.

The mean residue hydrophobicity plot pro-

vides an opportunity to visualize hydrophobic

weightings of sequences and to recognize

pattern similarities between chains that may be

associated with function, as seen with the

myoglobin and hemoglobin chains in Figures

7.2 and 7.9. Recall that the mean residue

hydrophobicity plot averages the value for an

11 residue sliding window with the numbered

residue as the central residue and with 37° C

being the value used entering and leaving the

sequence. For example, the mean value for

residue 1 uses TfValues of

37°

C for positions

1 to 5 and the averaged value of residues 1

through 6; and the mean Tt-value for residue 6

is the average value for the first 11 residues.

7.4.2.2.1 y-Fibrinogen—A Striking

Hydrophobic Sequence Near the

Carboxyl End

As shown in Figure 7.29, from the perspec-

tive of hydrophobic sequences, residues 337

through 379 of the y-fibrinogen chain consti-

tutes the most hydrophobic sequence, and the

mean residue hydrophobicity plot bears an

interesting pattern of hydrophobicity peaks

that bears a striking resemblance in shape and

intensity to the 401 to 448 sequence of the

BP-fibrinogen chain (Figure 7.30B). There are

additional mean residue hydrophobicity peaks

of lesser prominence centered near residues

240,

278, and 310. This provides an interesting

opportunity to look for correlations with points

of intermolecular association leading to fibrin

clot formation as represented by the crystal

structure data.

7.4.2.2.2 Aa-Fibrinogen—A Striking

Hydrophobic Sequence (-80 Residues)

at Midsequence

The most striking feature of the Aa-fibrinogen

mean residue hydrophobicity plot (plot not

included), a sequence of 610 residues, is the

central 81 residue sequence (residues 284 to

365) that is devoid of all but a single charged

residue, which is the moderate lysine (Lys, K)

residue and which contains four tryptophan

(Trp,

W) residues, the most hydrophobic

residue. The mean residue plot for Aa-

fibrinogen (plot not shown), however, is devoid

of significant hydrophobic peaks in contrast to

those observed in Figure 7.9 for the a- and P-

chains of hemoglobin A and for those of the y-

fibrinogen chain in Figure

7.29.

The presence of

the repetitive central sequence in humans, but

not in the chicken sequence, would provide a

striking opportunity for hydrophobic associa-

7.4 Blood Clotting: Poised at the Cusp of Insolubility

289

Human y-fibrinogen

Residue number

FIGURE 7.29. Single residue (A) and mean residue

(B) hydrophobicity plots of human y-fibrinogen. The

most striking feature of the mean residue plot is the

hydrophobic sequence, residues 337 to 379, experi-

mentally shown to provide the

site for interaction

with the a-chain amino-terminus on removal of

the

peptide and exposure of GPRP and its surrounding

hydrophobic residues for association with the

hydrophobic domain of the

site shown in Figure

7.32A. A similar situation obtains for the

chain

site,

as shown in Figure 7.32B. In our view, A and B

peptide removals lower the cusp of insolubility for

fibrin clot formation by making possible these

hydrophobic associations. Accordingly, blood clotting

occurs by means of

movable cusp of

insolubility,

the

central point being that hydrophobic association, low-

ering the cusp of insolubility,

central to aggregation

of molecules for blood clotting.

tion. This feature does not come into play in

the structures in Figures 7.31 and 7.32, nor do

the missing 372 amino-terminal residues of the

chicken sequence. TTiese differences w^ith

humans do not seem to interfere with carrying

the insight of the chicken structure to the frag-

ment DoubleD of human fibrin. Fortunately,

the human fibrin-DD was obtained with the

same bound GPRP and GHRP peptides, noted

above, the exposures of which trigger fibrin clot

formation.

7.4.2.2.3

BP-Fibrinogen—A Striking

Hydrophobic Sequence (-50 Residues) Near

the Carboxyl End

The mean residue hydrophobicity plot of BP-

fibrinogen in Figure

7.30B

exhibits two minor

hydrophobic sequences near residues 115 and

375 that could serve hydrophobic folding and

assembly within the fibrinogen molecule, but

would be less likely to participate in the

hydrophobic association between molecules.

290

Biology Thrives Near a Movable Cusp of Insolubility

200 250 300

Residue number

FIGURE 7.30. Single residue (A) and mean residue

(B) hydrophobicity plots of human Bp-fibrinogen.

The most striking feature of the mean residue

hydrophobic plot of this sequence is due to residues

401 to 446. The pattern of mean hydrophobicity

arising from residues

401

to 446 bears striking resem-

blance to that of residues 337 to 379 of y fibrinogen

(see Figure 7.29B) that have been defined as forming

the

site. Based on the pattern similarity with the

site,

residues

401

to 446 become recognized as the

site for hydrophobic association with site exposed

on removal of the B peptide at the central domain.

Accordingly, removal of the B peptide exposes

GHRP and its hydrophobic surroundings for

hydrophobic association with the P-chain EB site

shown in Figure 7.32B.

The most striking hydrophobic sequence of BP-

fibrinogen runs from residues 401 through 448,

and the pattern of the mean hydrophobicity

bears a striking resemblance to that of residues

337 through 379 of the y-fibrinogen chain.

The latter became a definition of the EA site

such that the pattern of mean hydrophobicity

of residues 401 through 448 of P-fibrinogen

becomes a definition of the

site. The actual

sites have now been determined by X-ray

dif-

fraction of a human fibrin fragment, and direct

observation of the sites is given in Figures 7.31

and 7.32.

7,4,2.3

Thrombin Activation of Fibrinogen

Provides for Hydrophobic Association to

Form the Soft Fibrin Clot

7.4.2.3.1 Removal of Peptide A (16 Residues)

Initiates Intermolecular Hydrophobic

Association

Removal of the first 16 residues of Aa-

fibrinogen activates fibrinogen for fibrin forma-

tion by removing the repulsive effect of four

negative charges of carboxylates, -COO", from

t^o glutamic acid (Glu, E) and two aspartic

7.4 Blood Clotting: Poised at the Cusp of Insolubility

291

acid (Asp, D) residues. This also decreases the

apolar-polar repulsive free energy of interac-

tion expressed in Equation (5.13) of Chapter 5,

section 5.7.9, and allows hydrophobic associa-

tion to proceed. The result is the exposure of

a GPRP sequence from each a-chain of the

central domain for association with

sites of

y-fibrinogen.

The EA site provides for association with

another fibrinogen molecule, that is, with the

(Aa)2(B (3)2(7)2 molecule. The experimentally

defined sequence that effects polymerization of

end-to-end DD docl

R Eg site for CHRP

^ of P-N-terminus

sites for

GPRP

of a-N-termi

PRP

'^^i:^*

[# ^.

FIGURE 7.31. Human fibrin-DD association sites

with residues given as aromatics (black), other

hydrophobics (gray), neutrals (light gray), and

acidics and basics (white). (A) One side of end-to-

end docking of D fragments, with D on the left side

in ribbon representation and D on the right side in

space-filling representation. (B) Opposite side of

end-to-end docking of D fragments, with D on the

left side in ribbon representation and D on the right

side in space-filling representation. The latter shows

both

sites that would interact with the two GPRP

sequences exposed at the central domain on removal

of the A peptides by thrombin to reveal

site,

iden-

tified here by bound GPRP tetrapeptide. (C) Stereo

view of one end of end-to-end DD docking

site.

Only

about one-half of the area shown is actually contact

area, and it is of modest hydrophobicity,

AGHA

~ -8

kcal/mole-contact surface. (Prepared using the crys-

tallographic results of Everse et al.^^ as obtained

from the Protein Data Bank, Structure File IFZC.)

292

Biology Thrives Near a Movable Cusp of Insolubility

FIGURE

7.32.

Stereo views of human fibrin-DD asso-

ciation sites with residues given as aromatics (black),

other hydrophobics (gray), neutrals (light gray), and

acidics and basics (white). (A)

site (with bound

GPRP tetrapeptide), at y-globular prominence with

an estimated

AGHA

of -16kcal/mole-surface, indicat-

ing a compelling globular prominence for hydropho-

bic association, especially when doubled as occurs in

the end-to-end docked structure. (B)

site (with

bound GHRP tetrapeptide), at p-globular promi-

nence with an estimated AGHA of -8kcal/mole of an

approximated contact surface area indicating a mod-

estly hydrophobic globular prominence for associa-

tion. (Prepared using the crystallographic results of

Everse et al.^^ as obtained from the Protein Data

Bank, Structure File IFZC.)

fibrin by association with the EA site is

sequence 337 to 379 of y-fibrinogen.^^ The poly-

merization sites are delineated in Table 7.1,

which gives the sequences. As shown in Figure

7.29B

and as noted above, this is the most

striking hydrophobic sequence of y-fibrinogen.

Accordingly, by simple inspection of the mean

hydrophobicity plot for y-fibrinogen, we see

the activation of fibrinogen as the emergence

of a site for hydrophobic association with

the most hydrophobic sequence in a second

molecule.

7.4 Blood Clotting: Poised at the Cusp of Insolubility

293

Furthermore, as seen in Figure 7.28, the EA

site is approximately at right angles to and

shares a common edge with the end-to-end

DoubleD association site such that these sites

could cooperate in initiation of the hydropho-

bic associations for soft fibrin clot formation.

7.4.2.3.2 Removal of Peptide B (14 Residues)

Allows for Further Intermolecular

Hydrophobic Association

Removal of the first 14 residues of Bp-

fibrinogen activates fibrinogen for fibrin forma-

tion by removing the repulsive effect of three

negative charges of carboxylates, -COO", from

two glutamic acid (Glu, E) residues and one

aspartic acid (Asp, D) residue. Removal of the

B peptide creates or exposes a GHRP site near

the central domain defined in Figure 7.27 that

associates with the carboxyl-terminal end of

the p-fibrinogen, the EB site, chain of another

fibrinogen molecule.^^ This sequence has not

been specifically delineated as above for y-

fibrinogen. Nonetheless, based on the structural

homology between y-fibrinogen and p-

fibrinogen, as demonstrated by the P-chain and

y-chain topologies given in Figure 3a of Sprag-

gon et

al.,^"*

and particularly based on the mean

residue plot of the y-chain (Figure 7.29B) with

its striking similarity to the mean residue plot

of the p-chain (Figure 7.30B), the correspond-

ing sequence for association with the EB site

would involve residues within the sequence

from residues 401 through 448. The mean

residue hydrophobicity plot for residues 401

through 448 in B P-fibrinogen demonstrates

hydrophobic homology to sequences 337 to 379

of y-fibrinogen (Figure 7.29B). Accordingly, by

simple inspection of a mean hydrophobicity

plot, this time for P-fibrinogen, we again see a

dominantly hydrophobic sequence and recog-

nize hydrophobic association as the basis for

soft fibrin clot formation.

7.4.2,4

Hydrophobic Associations of

Fragment DoubleD from Human Fibrin

When the fibrin clot is cleaved by the protease

plasmin, it fragments into two parts, a D frag-

ment and a smaller E fragment. Although no

crystal structures have been obtained for the E

fragment, DooUttle and coworkers have

obtained crystal structures for a factor XIII

(thrombin-activated transglutaminase) cross-

linked DoubleD fragment in which D frag-

ments are attached end to end.^"^ Fortunately,

these crystal have been obtained and the struc-

tures solved with bound GPRP and GHRP

peptides such that the EA and EB sites have

been identified.^^ With these accomplishments

of the Doolittle group, it becomes possible to

characterize the associations of chains that

result in blood clotting. The objective is to

assess the hydrophobicity of the association

sites to challenge further our perspective that

"biology thrives at the cusp of insolubiUty,"

which cusp of insolubility represents the

process of hydrophobic association.

7.4.2.4.1 Hydrophobicity of the Points

of Association

Three key points of association become clear.

They are the EA and

sites identified on the

fibrinogen molecule of Figures 7.27B and 7.28

and demonstrated in the human fragment

DoubleD of Figure 7.31 A,B and the end-to-end

docking site found in the human fragment

DoubleD. This provides the opportunity to

examine directly the hydrophobicity of these

three sites of aggregation for soft fibrin clot for-

mation and to estimate hydrophobicity in terms

of estimates of relative values for the Gibbs

free energy of hydrophobic association, AGHA,

of the contact faces or prominences.

7.4.2.4.2 End-to-End Docking Uses the

Carboxyl-terminal Globular Component

of the y-chain

With the crystal structure, the end-to-end

docking prominence becomes defined. The

docking site is shown in side views in Figure

7.31A,B and end on in Figure 7.31C. This is a

site of modest hydrophobicity that involves

using only about half of the end observed in

Figure 7.31C. One estimates the Gibbs free

energy of hydrophobic association,

AGHA,

be -8kcal/mole of contact surface. It should

be appreciated that this is a gross estimate

that should be improved once appropriate

programs have been prepared. The calculation

294

Biology Thrives Near a Movable Cusp of Insolubility

utilizes the values,

AGHA,

in Table 5.3 for the

amino acid residues and subjectively reduces

those values with crude estimates of the

amount of exposure of the side chain to the

surface concerned and a further reduction of

the contributions of charged residues by half

when ion paired.

7.4.2.4.3 Hydrophobicity of the

Site and

a Presumed Cooperative Binding with

End-to-End Docking

As is apparent in Figure 7.31B, the EA site

occurs as a double site at the junction of two

molecules. Coarse calculations of the free

energy for hydrophobic association at each of

the EA sites in Figure 7.32A indicate an espe-

cially hydrophobic surface with an estimate for

AGHA

of -16kcal/mole-EA site, which would be

twice that number for the double site. It seems

reasonable to expect that two sites of the

fibrinogen molecule exposed at the central

domain on removal of the A peptides from the

amino terminus of each a-chain would them-

selves cooperatively bind with both EA sites.

The process of assembly of the two

sites on

different fibrinogen molecules with the two

thrombin exposed GPRP-containing sites in

proper relationship at the central domain could

be expected to cooperatively facilitate the end-

to-end docking. In this way thrombin activation

of fibrinogen would initiate fibrin clot forma-

tion. Initiation of fibrin clot formation becomes

the stitching together of hydrophobic domains.

"Biology thrives at the movable cusp of

insolubiUty...."

7.4.2.4.4 Hydrophobicity of the Eg Site

The

site gains its potential for hydrophobic

association primarily by the unusual clustering

of the most hydrophobic residue, tryptophan,

specifically residues W424, W440, W444, and

W385,

as well as the additional very hydropho-

bic aromatic residues F374, F457, Y345, and

Y445.

Five of those residues contribute to the

mean residue hydrophobicity pattern in Figure

7.30B, and small adjustments in surface expo-

sure on association could create the most

hydrophobic single site for fibrin assembly.

Perhaps the 272 missing carboxyl-terminal

residues of the a-chains serve to cover the

hydrophobic

and

sites until pushed aside

by the much greater hydrophobic association

potential of the sites exposed on removal of the

A and B peptides.

7.4.2.4.5 The Limited Information on

the GPRP and CHRP Sites at the

Central Domain

The missing 96 residues at the amino termini of

the three chains, in the vicinity of the central

domain, preclude definition of the hydrophobic

domains that pair with the

and

sites, but

one predicts the capacity of these missing

residues to arrange in formation of hydropho-

bic domains with which to pair with the total of

four

and

sites.

A description of the general conclusion of

blood clotting as driven by hydrophobic associ-

ation follows.

7.4,2,5

Cross-linking to Form the Hard

Fibrin Clot

The hydrophobic associations with the EA site

of the hydrophobic 337 to 379 sequence of the

y-chain and with the

site of the hydrophobic

409 to 451 sequence of the p-chain result in

antiparallel alignment of the y-chains from

dif-

ferent fibrin molecules near the end-to-end

y-chain-y-chain docking site. In particular,

by means of a transamidase, glutamine-399 of

one y-chain cross-links to the lysine-406 of the

abutting second y-chain, and glutamine-399 of

the second y-chain cross-Unks with the

lysine-406 of the first y-chain to form two £-(y-

glutamyl)lysine cross-links. These chemical

cross-links transform the soft fibrin clot irre-

versibly into the hard fibrin clot, which can only

be disassembled by subsequent proteolytic

enzymes.

ThuSy

the key process of fibrin clot formation

is one of cleaving peptide sequences, the result of

which is to lower the cusp of insolubility^ repre-

senting the hydrophobic association between

molecules, from above to below physiological

temperature.