Platelets, August 2009; 20(5): 349–356

ORIGINAL ARTICLE

Inhibition of c-thrombin-induced human platelet aggregation by histoneH1subtypes and H1.3 fragments

GERALD SOSLAU1,2, PHILLIP J. PREST2, REINER CLASS3,4, MONIKA JOST3,

& LYNN MATHEWS2

1Department of Biochemistry & Molecular Biology, 2Office of Professional Studies in the Health Sciences,3Department Radiation Oncology, Drexel University College of Medicine, Philadelphia, PA, USA, and4Toxicology & Premedical Development, Pharmacelsus, GmbH Science Park 2, 66123 Saarbrucken, Germany

(Received 12 March 2009; revised 1 May 2009; accepted 13 May 2009)

AbstractHuman platelets are differentially activated by varying concentrations of �-thrombin or by �- and g-thrombin viathree thrombin receptors, PAR-1, PAR-4 and GPIb ~�. It is likely that the development of a normal or abnormal hemostaticevent in humans is dictated, in part, by the selective activation of these receptors. The ability to differentially inhibitthese thrombin receptors could, therefore, have clinical significance. We have previously demonstrated that histoneH1 selectively inhibits the PAR-4 receptor. In the current study we investigated whether five subtypes of the H1 moleculeor fragments of the H1.3 subtype differentially inhibited the PAR-4 receptor. PAR-4 inhibition by all H1 subtypeswas saturated at 1 uM with no statistical difference observed with the five H1 subtypes tested. Of the five fragmentsgenerated from the H1.3 molecule only one had significant inhibitory activity against PAR-4. The C-terminal fragment,N.1, generated by the proteolysis of the parent molecule by Asp-N endoproteinase (Aeromonas proteolytica) at the singleaspartate residue, showed the same level of PAR-4 inhibition as the intact H1.3 at 1 uM concentrations. Removal of twoN-terminal amino acids (Asp-Val as determined by MALDI analysis) from the N.1 fragment further enhanced itsinhibitory activity. These studies may help to develop specific drugs to differentially inhibit the platelet thrombin receptors.

Keywords: Platelet aggregation, �–thrombin, histone 1

Introduction

Hemostasis and thrombosis are orchestrated by the

generation of �-thrombin, a serine protease, derived

from the inactive precursor, prothrombin (factor II).

Thrombin is required for both the intrinsic and

common coagulation cascades [1], generates the

proteolytic conversion of fibrinogen to fibrin

required for clot formation and also activates

platelets via specific thrombin receptors. Evidence

indicates that �-thrombin may be autolytically

cleaved, in vivo, to initially form �-thrombin which

in turn can be further hydrolyzed to yield g-thrombin

[2, 3]; the same process can be induced in vitro with

the addition of plasmin, factor Xa, or trypsin [2].

These additional forms of thrombin may be physi-

ologically significant by differentially activating three

human platelet thrombin receptors, PAR-1 [4],

PAR-4 [5], and GPIb [6, 7]. The mechanism of

thrombin activation of platelets via the protease

activated receptors, PAR-1 and PAR-4, has been

well established [8]. The mode of thrombin-induced

activation of platelets via GPIb has not been as well

resolved. While one group indicated that GPIb was

not involved in thrombin-induced platelet aggrega-

tion at all [9], the accumulating evidence demon-

strates that GPIb-thrombin interactions are directly

involved in platelet aggregation by a unique pathway

[6, 7, 10–12]. �-Thrombin activates platelets pri-

marily via PAR-1 and/or GPIb, while g-thrombin

and �-thrombin only function through PAR-4 [6, 13,

14]. The specificity of the platelet thrombin receptors

offers the potential to selectively inhibit responses for

both clinical and research purposes.

Correspondence: Gerald Soslau, Office of Professional Studies in the Health Sciences, Drexel University College of Medicine, 245 N. 15th Street, MS 344,

Philadelphia, PA 19102, USA. Tel: þ215-762-7831. Fax: þ215-762-7434. E-mail: gsoslau@drexelmed.edu

ISSN 0953–7104 print/ISSN 1369–1635 online � 2009 Informa Healthcare Ltd.

DOI: 10.1080/09537100903047745

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The structural interactions of histone H1 with

DNA are well documented. Eukaryotic DNA is

efficiently packaged into a relatively small nuclear

volume called chromatin [15]. Chromatin consists

of repeating nucleosomes: four histone proteins – two

each of H2A, H2B, H3, and H4 – form an octamer

around which 160–220 base pairs of genomic DNA

wrap [15]. The nucleosome further contains an

average of one lysine-rich linker histone, H1,

resulting in the ‘‘beads on a string’’ motif that can

be visualized by electron microscopy [16]. In the

absence of H1, chromatin unravels into unstable

linearized filaments, demonstrating the importance of

H1 in stabilizing the structure of the nucleosome [17].

The protective role of DNA structure by histone

H1 is but one of many biological roles ascribed to

this protein. Histone H1 has been shown to affect

several different cellular processes: directly causing

cytolysis [18]; reducing aerobic and anaerobic

metabolism [19]; altering cell morphology and con-

fluent density [20]; limiting DNA and RNA produc-

tion [21], and; one subtype, H1.2, has been shown to

trigger apoptosis [22]. Histone H1 also affects the

progress and outcome of the inflammatory response

by activating macrophages and neutrophils while also

regulating several leukocyte functions [23]. In addi-

tion, histone H1 appears to possess anti-neoplastic

properties, including suppressing tumor activity

in experimental mammary carcinoma [24] and anti-

tumor effects against leukemic cells [25]. The

origin of H1’s cytotoxicity may be related to its

similarity with proteins secreted by activated macro-

phages [26]. Furthermore, H1 directly activates

macrophages to have nonoxidative antimicrobial

potential; these activated macropages may recognize

and destroy neoplastic cells [27]. Histone H1 has

further been demonstrated to have strong antibiotic

properties, particularly against pathogenic Gram-

negative bacteria [28]. All histone H1 molecules

share the same three-domain structure demonstrat-

ing an unmatched degree of evolutionary conserva-

tion. Therefore, the secondary H1-structure seems to

be of major importance for its biological properties.

If histone H1, or fragments of H1 are systemically

administered into patients its effects on blood and

its components must be thoroughly understood.

The aim of the present study was to further expand

on our observation that histone H1 can inhibit

g-thrombin-induced platelet aggregation [14]. Here

we show that recombinant subtypes of H1 and one

proteolytic fragment of the H1.3 molecule all

selectively inhibit g-thrombin activation of human

platelets via the PAR-4 receptor. This selective

inhibition of the platelet aggregation – PAR-4 path-

way, especially by a fragment of the H1.3 molecule,

provides a basis for the potential development

of specific drugs to differentially inhibit platelet

thrombin receptors.

Materials and methods

Recombinant histone H1 subtypes (H1.0, H1.2,

H1.3, H1.4 and H1.5) were purchased from Alexis

Biochemicals (San Diego, CA, USA). Five different

fragments of the H1.3 subtype (N.1, N.2, NG, G,

GC) were prepared by enzymatic cleavage [29]. The

five H1.3 fragments refer to different regions of the

peptide (N-terminal, C-terminal, globular regions),

with N.1 and N.2 representing complementary

pieces resulting from a single proteolytic cut (see

Figure 1). Fragments N.1 and N.2 were prepared by

digesting rhH1.3 with endoproteinase AspN (Sigma,

St. Louis, USA), (2 U/mg protein) over night at 37�C

and purified by HPLC using a Vydac RP 214TP

column and eluted with water and acetonitrile.

The identification and characterization of the

proteolytically generated H1.3 fragments, N.1 and

N.2, was critical since the N.1 fragment is the only

peptide that significantly inhibited g-thrombin-

induced platelet aggregation. The peptide eluting

early from an HPLC column should be the lower

molecular weight N-terminal fragment (m wt 7009)

and the second peak should be the C-terminal

(15,230 m wt) peptide. However, when these HPLC

fractions were subjected to PAGE (polyacrylamide

gel electrophoresis) analysis the early eluting fraction

migrated slower as a single band and the second

fraction migrated faster as a lower molecular weight

band. This occurred with both a 15% gel and a

10–20% gel and neither fraction migrated at the

appropriate apparent molecular weight (data not

shown). Samples were therefore analysed by MALDI

mass spectrometry to correctly identify the proteoly-

tic fractions. The MALDI analysis was conducted by

the Washington University Mass Spectrometry

Service (St. Louis, MO, USA). MALDI analysis

clearly demonstrated that the early and late eluting

peaks from HPLC were indeed the N-terminal and

C-terminal peptides, respectively (Figure 2a, b). N.1

is designated the 15,237 C-terminal fragment while

N.2 is the 7009 N-terminal fragment. It is interesting

to note that the N-terminal fragment consists of two

species, one with no N-terminal methionine group

(m wt 7009 daltons) and one with 2 N-terminal

methionine groups (m wt 7272 daltons) in a 2:5

ratio. The second peak in Figure 2b at an apparent

m wt of 7612 daltons is actually a dimeric form of the

15,237 dalton species. This was proven by analysing

the sample by deconvolution of the electrospray mass

spectrometry data and by mass spectrometry of

tryptic digests. The final spectrum shows a single

peak at 15,229 daltons and a 7614 species that is an

artifact of the deconvolution program (Figure 2c).

The tryptic data is not shown.

Blood was obtained from donors who signed

consent forms under an approved IRB protocol, by

venipuncture and collected in 4.5 ml sodium citrate

vacutainers. Washed platelets were prepared as

350 G. Soslau et al.

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previously described [30] where the final

platelet pellet was gently disrupted with 10 ml of

pH 6.0 sodium citrate solution (1 mM sodium

citrate in 20 mM Tris, 150 mM NaCl) followed

by their resuspension in hepes Tyrode’s buffer,

(136 mM NaCl, 2.7 mM KCl, 3.3 mM NaH2PO4,

1.0 mM MgCl2, 3.8 mM Hepes, pH 7.4) with

2 mg/ml dextrose and 1mg/ml bovine serum albu-

min at the initial PRP (platelet rich plasma) volume

at 2 – 3� 105 platelets/ml.

Platelet aggregations were conducted on a dual

channel lumi aggregometer (Chronolog corp.

Haverford, PA, USA) with 480 ml of washed platelets

plus 5 ml human fibrinogen (final concentration of

100 mg/ml). Platelet samples were incubated for

2–4 min with the histones/histone fragments prior

to the addition of agonist. In some experiments the

histone fragments were pre-treated with N-terminal

aminopeptidase (AP) from Aeromonas proteolytica

(Sigma) for varying times prior to the addition to

Figure 2. MALDI mass spectrometric analysis of the histone H1.3 fragments N.1 and N.2. (a) Fragment N.2; Panel (b) fragment N.1;

Panel (c) spectrum of fragment N.1 after deconvolution of the electrospray mass spectrometric data; (d) fragment N.1 treated with

aminopeptidase as described in Methods.

Figure 1. Schematic of the structural regions of the histone H1.3 molecule and its fragments.

Inhibition of �-thrombin-induced human platelet aggregation by histone H1subtypes and H1.3 fragments 351

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washed platelets. Aggregations with these samples

were then conducted as above. In all cases when

g-thrombin was employed as the agonist the

concentration was experimentally set to yield

about 60–80% aggregation (final concentration of

g-thrombin 8–25 nM).

Histone H1.3 inhibition studies of thrombin

activity with the synthetic substrate, H-D-CHG-

Ala-Arg-pNA 2AcOH (Pefachrome TH, Cen-

terchem, Inc., Norwalk CT, USA), were conducted

as previously described [31]. The generation of a

colored product by the hydrolysis of the substrate in

the reaction buffer (50 mM Tris-HCL, 50 mM

imidazole, 150 mM NaCl, pH 8.0) was monitored

at 405 nm over a 4 min time period on a Hitachi

U-2000 spectrophotometer. Reactions were run in

triplicate with 10 and 20 nM g-thrombin or with

0.5 U/ml �-thrombin (Chronolog, Corp, Havertown,

PA, USA). The Pefachrome TH substrate concen-

trations employed were 0.05, 0.1 and 0.2 mM.

Results

We have previously demonstrated that histone H1

selectively inhibits g-thrombin – induced platelet

aggregation and has no effect on the �-thrombin –

induced reaction [14]. The current studies focused

on potential structural aspects of histone1 inhibition

of g-thrombin-induced platelet aggregation. Previous

studies were performed with tissue-derived bovine

histone H1, which represents a mixture of several H1

subtypes. At least seven different subtypes or variants

of H1 have been described in higher eukaryotes (for

review see [32]). The globular core domain is highly

conserved between the subtypes; whereas the N- and

C-terminal regions are more divergent, which may

result in functional differences. For example,

only the H1.2 subtype has been shown to trigger

apoptosis [22]. To further dissect the contribution of

different subtypes to inhibiting g-thrombin–induced

platelet aggregation, we compared several human

full-length H1 subtypes in a typical platelet aggrega-

tion assay. All 5 subtypes of histone H1 tested

inhibited g-thrombin–induced platelet aggregation

with no significant difference at 1mM (Figure 3). The

degree of inhibition of platelet aggregation was

dose dependent with no statistical difference in the

inhibitory activities between the subtypes tested

(Figure 4). For further studies, H1.3 was used to

generate fragments of the parent molecule.

Fragments of the histone H1.3 molecule, as depicted

in Methods, were tested to determine if the inhibi-

tory activity resided in a portion of the molecule or

required the intact molecule. Of the fragments

tested, only the N.1 fragment of histone H1.3

inhibited platelet aggregation (Figure 5). At the

concentration tested (1mM), inhibition was compa-

rable to the full-length H1.3 while the N.2 fragment

had little to no effect at the same concentration, and

sometimes appeared to slightly stimulate the reac-

tion. Other fragments, NG, GC, G alone, or in

combination with N.2, also had little or no effect at

1 mM concentrations (Figure 5). This suggests that

the inhibitory activity resides within the C-terminal

portion of the molecule and, possibly, the third helix

of the globular domain. However, since the GC

fragment had no effect, it has to be assumed that the

C-terminal domains within these two molecules (N.1

and GC) have different conformations, the C-

terminus of N.1 presumably being similar to that of

full-length H1.3.

Next we investigated whether adding the N.1

fragment to the non-inhibitory fragment N.2 would

reveal a regulatory role of inhibition to N.2, i.e.

whether the two parts of the protein could function

when separated into two different subunits. In these

assays, N.1 was added at 0.1, or 0.2 uM as opposed

to the maximum concentration tested (1 uM).

Gamma-thrombin inhibition

0.00 0.25 0.50 0.75 1.00 1.250

102030405060708090

100110

H1.0

H1.2

H1.3

H1.4

H1.5

Histone conc. (uM)

% In

hib

itio

n

Figure. 4. Dose-response of g–thrombin-induced platelet aggre-

gation inhibited with different histone 1 subtypes. The inhibition

of platelet aggregation was conducted with four different

concentrations of each H1 subtype, in triplicate, with the

meansþ/-standard deviations graphed. There was no statistical

difference between groups of data by an ANOVA and Kruskal-

Wallis test.

H1.0 H1.2 H1.3 H1.4 H1.560

70

80

90

100

Histone 1 subtype

% In

hib

itio

n

Figure 3. The inhibition of g–thrombin-induced platelet aggre-

gation by histone 1 subtypes. The final concentration of each

histone 1 subtype was 1mM and the bar represents the mean for

each subtype. Each point represents an individual experiment.

352 G. Soslau et al.

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Inhibition by N.1 alone was kept in the approxi-

mately 40% range by adjusting the concentration to

either 0.1 or 0.2 uM in order to determine if the

addition of N.2 significantly altered the final level of

inhibition. When N.1 was added to 1 mM N.2, which

by itself shows no inhibitory activity, inhibition

increased to about 75%, indicating a synergistic

effect of the two molecules. This appears to demon-

strate that the two fragments can function as subunits

and mimic the full-length protein (Figure 6).

Our previous studies indicated that H1 inhibited

g-thrombin-induced platelet aggregation at two

levels. There appeared to be a direct effect on

both the thrombin molecule and the PAR-4

receptor [14]. However, under the experimental

conditions described in Methods, no direct inhibi-

tion of �-, or g-thrombin could be detected using a

chromogenic peptide substrate for thrombin.

Therefore, independent of a direct effect on

g-thrombin, it was important to demonstrate that

the N.1 inhibition, like H1, again included a

specific direct effect on the PAR-4 receptor without

involving other agonist receptors. Figure 7 clearly

demonstrates that platelets, preincubated with

1.5 uM N.1 for 4 min prior to the addition of

agonist, were only inhibited at the PAR-4 receptor.

This inhibition was observed with either g-throm-

bin or TRAP-4A (AYPGKF). The reaction with

TRAP-4A was significantly inhibited by N.1 but

not as completely inhibited as was the g-thrombin

reaction.

Finally, we investigated the potential role of the

N-terminal residues of N.1 for platelet inhibition by

enzymatic removal of the N-terminus amino acids

with AP. The amount of AP employed for these

experiments was determined by titrating the amount

of enzyme added to platelets for 2 min prior to the

addition of g-thrombin. The maximum amount of

AP that resulted in no, or minimal levels of,

inhibition of platelet aggregation was employed. AP

concentrations above 0.1 U AP/ml significantly

inhibited the reaction with 1.0 U AP/ml being com-

pletely inhibitory. The random removal of

N-terminal amino acids of platelet surface proteins

with 1.0 U AP/ml also inhibited PAR-1-, collagen-

and ADP-induced platelet aggregations (data not

shown). Figure 8 shows a representative time course

N1 effect on aggregation

g-T PAR-1 PAR-4 U-4 Coll ADP0

25

50

75

100

Control

+1.5uM N1

Agonist

% A

gg

reg

atio

n

Figure. 7. The effect of N.1 on platelet aggregation induced by

various agonists. The platelets were either untreated (control) or

preincubated for 4 min with 1.5 uM N.1 followed by agonists at a

concentration sufficient to induce 60–80% aggregations. g-T is

g-thrombin (15 nM), PAR-1 activated with TRAP-1 (0.45 uM

SFLLRNP), PAR-4 activated with TRAP-4A (60 uM AYPGKF),

U-4 is the thromboxane (T�A2) analog, U46619 (50 uM), coll is

collagen (1–5 ug/ml), and ADP (1–10 uM). The ADP aggrega-

tions were conducted with PRP while all other aggregations were

with washed platelets. Each data point is a repeat of three separate

experiments with the meanþ/-standard deviations depicted. Only

the results with N.1 plus g-thrombin or TRAP-4A were

significantly different than their controls, as analyzed by a

student-t test.

Figure. 6. The synergistic inhibitory effect of histone H1.3

fragments N.1 plus N.2 on g-thrombin-induced platelet aggrega-

tion. The designation of the fragments are as in Figure 1. The final

concentration of N.1 employed was either 0.1 or 0.2 uM plus

1 uM N.2. Each point represents an individual experiment.

Figure 5. The inhibition of g–thrombin-induced platelet aggre-

gation by H1.3 fragments and combination of fragments

compared to full-length histone H1.3. The designation of the

fragments are the same as in Figure 1. All fragments were tested at

a final concentration of 1 uM. Each point represents an individual

experiment.

Inhibition of �-thrombin-induced human platelet aggregation by histone H1subtypes and H1.3 fragments 353

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experiment in which inhibition of g-thrombin-

induced platelet aggregation was measured after

digesting 0.1 mM N.1 with 0.075 U AP for

0–110 min. Platelets were preincubated with the

modified N.1 for 2 min prior to the addition of the

g-thrombin. Platelet receptors, other than PAR-4,

are not affected by 0.075U AP under these condi-

tions. Interestingly, the capacity of N.1 to inhibit

platelet aggregation increased with the first 10 min of

digestion time and then reached a plateau indicating

that any modification of N.1 by AP was completed by

about 10 min.

MALDI analysis of the AP-treated N.1 fragment

(5 ml 100 mM N.1 plus 1 U AP at 37�C for 90 min)

demonstrated that two N-terminal amino acids (Asp-

Val) were hydrolyzed off and the reaction did not go

any further (Figure 2b, d). This conclusion was

based upon the difference between the digested and

undigested N.1 fragment, which was about 212

daltons (the molecular weight of peptidyl-linked

Asp-Val amino acids), resulting in the 15,025

dalton fragment. Removal of these two amino acids

appears to impact on the conformation of the peptide

resulting in a more active molecule by maintaining

the peptide in a conformation with greater inhibitory

function.

Discussion

The presence of three different thrombin receptors

on human platelets that differentially respond to

varied concentrations of �-thrombin and to �- versus

�- and g-thrombin may have important physiological

regulatory roles in normal and abnormal hemostasis

[6, 13, 14]. We have extended our earlier studies that

demonstrated that histone 1 selectively inhibits

human platelet PAR-4 at the membrane level [14]

by pinpointing the region of one histone H1 subtype

responsible for inhibitory activity to the C-terminal

portion of the molecule. This may serve as a

blueprint for generating therapeutic molecules for

treatment of abnormal hemostasis.

We found that all five subtypes of the recombinant

H1 protein tested were approximately equally active

as selective inhibitors of PAR-4. We further dissected

one subtype, recombinant human H1.3, with respect

to functional domains by creating several fragments

by enzymatic cleavage. Of all fragments tested, only

the C-terminal 15 k N.1 fragment retained inhibitory

activity. This fragment is composed of the complete

C-terminus and the third helix of the globular

domain. The fact that another fragment, GC,

which includes the C-terminus and the whole glob-

ular domain, did not have significant inhibitory

activity, suggests that the other two helices of the

globular domain disrupt the conformation of the

C-terminus required for functional activity. It is also

possible that these two other helical regions are

exposed in such a way to prevent the third helix-C-

terminal structure, found in N.1, from interacting

with the PAR-4 receptor. The conformation and/or

amino acid sequence of the third helix may also have

a slight impact on the overall conformation or

binding properties of N.1, since removal of the two

N-terminal amino acids of the N.1 fragment further

increased its inhibitory activity.

The N-terminal amino peptidase (AP) from

Aeromonas proteolytica generally will not hydrolyze

aspartic or glutamic residues. The fact that the newly

generated N-terminal aspartate is hydrolyzed off

from the N.1 fragment indicates that it may have

been modified during hydrolysis of the parent H1.3

molecule. The AP was then able to rapidly hydrolyze

the valine residue and stopped at the newly generated

N-terminal glutamic acid residue which is resistant to

further hydrolysis. The alteration of the N-terminal

region of N.1 seems to play a significant role in

increasing the inhibitory potential of the H1.3

fragment. However, any structural modification in

the N.1 fragment due to removal of the Asp-Val that

may have occurred could not be detected by circular

dichroism (data not shown), which would indicate

little, or no alteration in the tertiary structural

elements. The C-terminal N.1 fragment contains

the amphipathic helix III region of the globular

domain and the unstructured C-terminal tail. The

helix III region is thought to be the major DNA

binding region of the H1 molecule. Also, the

‘‘unstructured’’ C-terminal region appears to be

able to assume �-helical structures in the appropriate

environment (such as the electrostatic environment

of DNA) [15]. It may be that these properties

facilitate binding of the N.1 fragment to the

platelet membrane/PAR-4 receptor as well as to the

g-thrombin molecule. As noted in our previous

publication [14], the crystalline structure and bind-

ing properties of �-thrombin and g-thrombin are

Figure. 8. The inhibition of g-thrombin-induced platelet aggre-

gation by the histone H1.3 fragment N.1 pre-treated with

aminopeptidase for varying times. This is a representative exper-

iment with 0.1 uM N.1 pre-treated with 0.075 U aminopeptidase

for 0–110 min (‘‘Time’’ on X-axis) at 37�C.

354 G. Soslau et al.

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quite distinct and likely account for some, or all of

the observed differential inhibition by N.1 of these

two forms of thrombin.

A second DNA binding region resides in the

globular domain associated with the N-terminal

fragment N.2 [15]. While there is little or no

inhibitory effect of N.2 by itself, it is interesting

that the addition of low levels of N.1 plus N.2 result

in a synergistic inhibitory response. This was not

observed with combinations of N.2 plus other H1.3

fragments. It is possible that the synergistic response

results from a conformational change induced by the

association of a region of N.2 with N.1 that increases

the exposure of the N.1 binding site for PAR-4 or a

membrane region near PAR-4 and/or a region on the

g-thrombin molecule. This enhanced binding may

mimic conformational changes of N.1 induced by AP

treatment that removes a charged Asp and a

lipophilic Val group.

It is clear that the N.1 fragment, like its parent H1

molecule, inhibits g-thrombin-induced platelet

aggregation at the PAR-4 receptor level. This con-

clusion is based upon the almost complete inhibition

by N.1 of the TRAP-4A-induced platelet aggregation

reaction. However, as previously observed [14],

the TRAP-4A reaction was not as fully sensitive as

the g-thrombin reaction and may indicate that N.1

is inhibiting aggregation by interacting both with the

g-thrombin molecule and the PAR-4 receptor.

However, a direct effect of H1.3 on the thrombin

molecule is not indicated by our studies with a

chromogenic thrombin substrate. It is possible that

there is, in fact, a small direct H1.3/N.1 effect on

thrombin that can not be detected by this system. A

small substrate molecule may still be able to interact

with the catalytic site on thrombin while interactions

with PAR-4 would be spatially blocked by the large

H1.3/N.1 bound to/associated with the thrombin

molecule. Inhibition was specific for the PAR-4

receptor and N.1 had no significant effect on any of

the other platelet receptors tested. The slight inhibi-

tion at the ADP receptor previously observed with

very high levels (10�) of H1(14) were not detected

here since platelets were preincubated with N.1 for

only 4 min versus the 30 min in our previous studies

and the high concentrations of N.1 compared to H1

were not used. The 4 min time interval was found to

give maximal levels of inhibition.

Much work remains to fully understand what

portion(s) of the H1 molecule selectively binds

to, and inhibits the human PAR-4 receptor and/or

the g-thrombin molecule. It would also be interesting

to compare the effect of N.1 fragments generated

from different H1 subtypes. It is possible that the

variable C-terminal region modifies/regulates the

binding/inhibitory potential of the ‘‘invariant’’ third

helical region. It is unlikely that H1, released into the

circulation from degraded cells, would reach a

sufficient concentration to have a significant clinical

impact on hemostasis. However, the full elucidation

of what sequence(s) of the molecule selectively binds

to and inhibits g-thrombin-induced platelet aggrega-

tion at PAR-4 may serve as a paradigm for the

production of drugs that have clinical significance in

regulating hemostasis at this receptor site.

Declaration of interest: The authors report no

conflicts of interest. The authors alone are respon-

sible for the content and writing of the paper.

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