Comparison of the primary structure of the functional domains of human and porcine von Willebrand factor that mediate platelet adhesion

Vol. 182, No. 2, 1992 January 31, 1992

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 561-568

Comparison of the Primary Structure of the Functional Domains of Human Porcine von Willebrand Factor that Mediate Platelet Adhesion

and

Bruce R. Bahnak, Jean-Maurfce Lavergne, Valerie Ferreira, Daniele Kerbiriou-Nabfas and Dominique Meyer INSERM U. 143 Hopital de Bicetre, 94270 le Kremlin-Bicetre, Received

November

20,

France

1991

Summary: Porcine von Wrllebrand factor (vWF) directly aggregates human platelets in vitro indicating a conformational difference between the human and porcine molecules. We amplified and directly sequenced 1242 nucleotides of porcine vWF cDNA that encodes functional domains which mediate the binding of vWF to platelets and subenclothelium. The deduced amino acid sequence corresponds to residues 473691 of the human mature vWF subunit and is 79% homologous with the human protein. Significant differences are found in two discontinuous segments thought to be involved in the binding of vWF to platelet glycoprotein lb. Porcine vWF lacks four contiguous residues in the first segment and has two positively charged arginine residues in the second. Three point mutations associated with human type IIB von Willebrand disease in the first segment of a botrocetin binding site are at the same position as mismatches between the pig and human. The second segment of the botrocetin site is highly conserved while the third segment shows only a 60% homology. 0 1992 Academic Press, Inc.

von Willebrand factor (vWF) is a complex multimenc glycoprotein which has a critical function as a mediator

of platelet

Circulating probably

adhesion

to the vascular

subendothelium

and as a carrier

human vWF does not bind to the platelet receptor glycoprotein requires a conformational

lb (GP lb). This interaction

change in vWF induced by its binding to the subendothelial

(2). The antibiotic ristocetin and a constituent of a snake (Bothropsjararaca) the action of vWF with the exposed endothelium (3,4).

There is, however,

a considerable

(6) suggesting a difference in the native conformation Determination

matrix

venom, botrocetin, mimic

and promotes the binding of human vWF to platelets

diversity in the response

ristocetin (4,5). In addition, porcine and bovine vWF spontaneously in the primary structure.

for factor VIII (1).

of vWF from various species to aggregate

human platelets in vitro

of vWF between species that should be reflected

of these differences

should aid in the understanding

of vWF

interaction with platelets and the subendothelium. We amplified corresponding functional

and directly

sequenced

1242

nucleotides

of the

to amino acids 473 to 891 of the human mature vWF subunit.

porcine

vWF cDNA

This area encompasses

domains of vWF involved in GP lb, collagen and heparin binding (1) as well as a proposed

Abbreviations:

vWF, von Willebrand

factor; vWD, von Willebrand

disease; GP, glycoprotein;

PCR,

polymerase chain reaction. 0006-291X/92

561

$1.50

Copyrighr 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

182, No. 2, 1992

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cleavage

BIOCHEMICAL

site associated

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

with the turnover of the molecule

(7). Specific differences

and

similarities in the amino acid sequence of human and porcine vWF are emphasized. Materials

and

Methods

Reverse Transcri~fase. RNA was isolated from porcine lung by a guanidinium isothiocyanate procedure (8). 2.5 ug of total RNA and 20 pmol of the appropriate primer (Fig.1) in a final volume of 8 ul were heated to 72°C and slowly cooled to 46°C. The reverse trancriptase reaction was in a 25 ul volume containing 5 ul of 5 x in vitro transcription buffer (Stratagene Inc., San Diego, CA), 1 mM of each dNTP, 25 units of RNasin (Boehringer-Mannheim, Mannheim, FRG). Avian myeloblastosis virus reverse transcriptase (15-20 units) (GenofiLGrand-Lancy, France) was added and the mixture incubated for 30 tin at 46°C. Polymerase Chain Reaction. 5 ul of the reverse trancriptase reaction were amplified in a 100 ul reaction volume containing 10 ul of 10 x DNA synthesis buffer (Amersham Inc., Amersham, UK), 100 uM of each dNTP and 0.5 uM of the oligonucleotide primer. The mixture was preheated at 94°C for 5 min before addition of 2 units of Taq polymerase (Amersham). The samples were amplified for 30 cycles of 1 min at 94”C, 1 min at 60°C and 1 min at 72°C before a final elongation step of 7 n-tin at 72°C. The products of the polymerase chain reaction (PCR) (9) were chloroform extracted, EtOH precipitated and etectrophoresed in a 3.0% composite agarose gel containing 2.5% low melting point agarose (NuSieve GTG, FMC Bioproducts, Rockland, ME) and 0.5% regular agarose (Seakem GTG, FMC Bioproducts) in TAE buffer (40 mM Tris-acetate, pH 8.0,2 mM EDTA) poured and run at 4°C. The band of the predicted size was cut from the gel and this agarose plug served as the stock for further amplifications. Direct Sequencing of the Amplified Products. Double-stranded templates for sequencing were produced using the same PCR procedure described above with 5 ul of the melted gel slice. Singlestranded template was generated by reducing the concentration of one primer to 50 r&I. The products from the second amplification were chloroform extracted, EtOH precipitated and electrophoresed as described above. The band was excised from the gel and placed in 1.5 ml of TAE buffer, centrifuged at 45,000 rpm (20°C) in a Ti 50 rotor for 30 min and the supernatant concentrated in a Centricon 30 column (Amicon Corp., Danvers, MA). The concentration of the DNA was estimated on a 3% agarose gel against a HAE Ill digest of 0x174 RF DNA molecular weight standard (New England Biolabs, Beverly, MA). Approximately 0.25 - 0.5 pmol of DNA were used for direct double-stranded sequencing (10) with a Sequenase kit (US Biochemical, Cleveland, OH). The DNA was EtOH precipitated, washed twice in 70% EtOH and dissolved in a volume of 11 ul containing 2 ul reaction buffer and 10 to 20 pmol of primer (40 to 1 ratio of primer to template). The DNA was heated at 100°C for 5 min, placed on dry ice, and centrifuged briefly before the addition of the labeling mix. Labeling reactions were for 1 min and termination reactions were for 2 min. For sequencing single-stranded templates, approximately 0.25 pmol of DNA (as estimated against double stranded molecular weight markers ) were EtOH precipitated as described above and mixed with 1 pmol primer. The conditions for annealing and sequencing single stranded DNA were essentially the same as recommended for the Sequenase kit.

465

D,H

Al

AZ

I

1909

3681

4960

33-34

53 -w 31 M-2

z

2 38

1 -e

3f

54

35 :

39 *

40

&& Amplification and sequencing strategy for a segment of porcine vWF cDNA. The black bar represents the nucleotide length of the amplified area. The nucleotide position is based on the sequence of Bonthron et al. (11) for human vWF cDNA with residue 1 the first nucleotide of the ATG initiation codon. The numbers below the bar refer to the oligonucleotides used for reverse transcriptase, amplification and sequencing (Table 1). The arrows indicate the orientation of the primers and the thin lines the length of the amplified fragments. The open bar represents the corresponding amino acids in the human mature vWF subunit and repetitive domains.

562

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Results and Discussion Porcine and human vWf is highly conserved. sequencing

Figure 1 shows the strategy for amplification

of the porcine vWF cDNA. The primers used (Table 1) were designed

exon 28 in the human gene with no thought to degenerate porcine sequence

contained

residues corresponding

1242 nucleotides

codons or third base pair wobble.

(Fig. 2). The deduced

to 419 amino acids in the human sequence

and

for the analysis of

amino acid sequence

The

was 414

(Ala- 473 to Phe-891) (Fig. 3). It

includes the C-terminal end of the D3 domain, the Al and most of the A2 repetitive domains of human vWF (12). There were five amino acids lacking in porcine vWF when compared to the human sequence including

four contiguous

homologous

residues

478 to 481.

The porcine

and human

sequences

were 79%

and 28 of the 89 mismatches were highly conserved amino acid changes.

Several

stretches of amino acids were well conserved

(Figs. 3 and 4).

A 76 amino acid

sequence from Arg-552 to Pro-627 was 90% identical and included a sulfatide binding domain (Lys-569 to Val-584) (13), the second segment of a botrocetin

binding site (Lys-569 to Gln-583)

(14) and was

within the limits of one of the collagen (amino acids 542 to 622) and one of the heparin (residues 512 to 673) binding domains (1). Amino acids 639 to 676 were 92% conserved segment of the botrocetin binding site (Arg-629 to Lys-643)

and included

part of the third

and the C-terminal boundary

of a heparin

binding domain. Significant

differences exist in the putative GP Ib binding domain. In vitro the function of human

vWF is assayed by its capacity to agglutinate ristocetin or botrocetin botrocetin, agglutinates

however,

indicating modulate

platelets in the presence of the non-physiological

that circulating differently

vWF does not interact with GP lb. Ristocetin and

the domains

on vWF and on GP lb (15,16). Porcine vWF

human platelets directly in the absence of exogenous

Table

1. Sequence

agonists

modulators

and Nucleotide Location of the Primers used for Reverse Amplification and Sequencing of the Porcine vWF cDNA

(6) suggesting

Transcriptase,

Primer

sequenCe(S-31

Location

pW2 pW3 pW33 pW34 pW35 pW37

CTGGAGGAGCCATCCAGCAGG ’ GTGTGGATGAGCTGGAGCAGCA 2 CACTCTAGATGlTGTCAACCTCACCTGTGAA2 -GCTGGCAATGCGCCGCAGCTCTGATG CACAAGClTCAGCGAGGCACAGTCCAAAGGGGGGA

pW39 pW40 pW53 pW54

GTGGTCAGAGAGGTACCGCAGGGCCAGC GGCCCACTCCAATGGGCACCA 3 GATRXGCGGATGGATGTGG 2 -GCAGCACCAGGTCAGGAGCC

3840-3880 4325-4348 3682-3705 401 O-4035 4653-4679 3928-3953 4488-4512 4737-4762 4874-4894 4572-4591 4980-4999

3 ’

-TCCCAGAAGTGGGTCCGCGTGGCCGT pW38 MCGAAlTCCAGGACGAACGCCACATCCAGA 3

3

2 3

that the

The location of the primers is based on the human vWF cDNA sequence (11). 1 Primer used for sequencing; 2primer used for amplification and sequencing; and 3primer used for reverse transcriptase, amplification and sequencing. The orientation of the primers is given in Figure 1. NonvWF sequences are underlined and contain engineered restriction enzyme sites for subcloning or a 51 base G+C-rich region (pW 54) on the 5’ end for denaturing gradient gel electrophoresis used to study the human vWF gene.

563

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GCCTGCGCGGAACCG************GTGCCCCCCACAGAAGGCCCGGCCCGGTCAGCCCCACCACACCCTACGAG ACAEP----VPP TEGPVSP T T

3718 497

P

YE

A

S

GAGGACACGCCAGAGCCGCCGCTGCACGACTTCTTCTTCTGCAGC~CTTCTGGACCTGGTCTTCCTGCTGGAC CSKLLD E D T PEPPLHDFF LVFLLD

3850 GGCTCTGATAAGCTGTCCGAGGCCGACTTCGAGGCCCTGACACCTG EALKVFVVGMMEHL 521G S D K L S E A D F 3922 545

CACATCTCCCAGAAGCACATCCGCGTGGCGGTGGTGGAGTACCACGACGGCTCCCACGCCTACATCTCGCTC RVAVVEYHDGS HAYISL H I S Q K H I

3994 569

CAGGACCGAAAGCGGCCCTCGGAGCTGCGGCGCATCGCCAGCCAGGTG~GTACGCG~CA~CAGGTG~T Q D R K R P S E LRRIASQVKYAGRQVA

4066 593

TCCATCAGCCAGGTTTTCAAGTACACGCTCTTCCAAATCTGCCTCCCGT S I S E V F K Y T L F Q I F

4138 617

ATAGCCCTGCTGCTCATGGCCAGCCAGGAGCCACGCCGGCTGGCCCAG~CTTGGCCCGCTACCTCCAGGGC I ALL LMA S Q E P RR LA QN LA RY

L

QG

4210 CTGAAGAAGAAGAAGGTCACCGTGATTCCGGTGGGCATCGGACCCCACGTCAGCCTC~GCAGATCCGCCTC 641L K KK KV T V I P VG I G P HV S L KQ

I

R

G

RV

D

R

P

E

4282 665

ATCGAGAAGCAGGCCCCGGAGAACAAAGCCTTTTGTGGTCAGCGGTGTGGACGAGCTGGAGCAGCGC~G~C I E KQAP E NKA FVV S GVD E L E

Q

4354 689

GAGATCATCAGCTACCTCTGCGACCTCGCCCCGGAAGTGC E I I S Y L C D LAP EVP

LVA

4426 713

GTCACTGTGGCGCCTGAGCTCCCCGGGGTTTCAACGCTCGCTCG~CCC~G~G***AGAATGGTCTTGGATGTG VT VA P E L P GV S T L E P K K RMVLDV

4498 737

GTGTTTGTGCTGGAAGGGTCCGACAAGGTCGGTCGGCCA VFV L E G S D KV G

EAN

A

F

P

N

T

R

RRP

S

T

E

F

L

RKN

Q

V

E

4570 GTGATCCGGCGGATGGACGTGGGCCGGGACAGTGTCCACGTCACGGTGCTGCAGTACTCGTACGTGGTGGCC 761V I RRMDV GRD S VHV TV L QY S

YVVA

4642 785

GTGGAGCACTCCTTCAGGGAGGCGCAGTCCAAGGGGGG~GTCCTACAGCGGGTGCGGGAGATCCGCTTCCAG VE H S F RE AQ S KG EV L Q RVR E

I

RF

4714 a09

GGTGGCAACAGGACCAACACTGGGCTGGCCCTGCAGTACCTCTCGGAGCACAGCTTCTCAGCCAGCCAGGGG G G N R T N T G LA L Q T L S E H S F S

A

S

Q

4786 833

GACCGGGAGGAGGCGCCCAACCTGGTCTACATGGTCACAGG~CCCTGCCTCGGATGAGATC~GCGGATG D RE EAP N LVYMV T GN P A S DE

I

K

RM

4858 857

CCGGGAGACATCCAGGTGGTGCCCATCGGGGTGGGCCCCGACGTGGAGATGCAGGAGCTAGAGCGCCTCAGC P GD I QVV P I GV G P D V E MQ E L E

R

L

4930 TGGCCCAATGCCCCCATCTTCATCCAGGACTTT 881WP NAP I F I QD

R

E

Q

G

S

F

E&2. Nucleolide sequence of the porcine vWF cDNA fragment and deduced amino acid sequence of the protein. The position of nucleotide residues is based on the human sequence (11). The amino acid sequence is given below and the position is based on the sequence of the human mature vWF subunit. Spaces are inserted in the nucleotide (‘) and amino acid (-) sequences to maintain the alignment with the numbering system for human vWF cDNA and protein.

native conformation

of porcine vWF has appreciable

affinity for human GP lb. Whether this conformation

mimics that induced by botrocetin or ristocetin is unknown. The porcine system responds to botrocetin and ristocetin, although weakly with the latter agent (5). Two discontinuous juxtaposed

segments (Cys-474 to Pro468 and Leu-694 to Pro-706) of the vWF subunit

by a disulfide loop formed by Cys-509 and Cys-695 are thought to interact with the platelet

receptor GP lb (17). Interestingly, these two short segments are within areas of porcine vWF that show significant differences

with human vWF (Figs. 3 and 4). A preliminary 564

description

of the partial bovine

Vol.

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BIOCHEMICAL

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H 473 P

GP Ib . . *A 9 GP Ib ACQEPGGLWPPTDAPVSPTTLYVEDISEPPLHDFYCSRLLDLVFLLDGSSRLSEAEFEV --A--****----EG------p-E--Tp--K-----------DK----D--A

H 533

........ . .... . ..Bot.................. LKAFVVD MMERLRISQKWVRVA~YHDGSHAYIGLKDRKRPSELRRIASQVKYAGSQVA . ..~

P

. . . . . . . . . Bat

...... l

.

.

......

--V---G---H-H----HI---------------S-Q-------------------R---

H 593 P

. . STSEVLKYTLFQIFSKIDRPEASRIALLLMASQEPQRMSRNFVRYVQGLKKK -I---F--------GRV------------------R-LAQ--L--------T-----

H 653 P

. . 0-m IGPHANLKQIRLIEKQAPENKAFVLSSVDELEQQRDEIVSYLCDLAFEAPPPTLPPDMAQ ----VS------------------V-G----RXN--I---------V-A--RR-LV--

H 713 P

. . . VTVGPGLLGVSTLGPKRNSMVLDVAFVLEGSDKIGEADFNRSKEF~EVIQRMDVGQDSI ---A-E-P-----E--K*R-----V--------V---N----T--V----R-----R--v

1..

- . . . . . . . ..Bot...

l

.. . . . . .......

.

. . . . HVTVLQYSYMVTVEYPFSEAQSKGDILQRVREIRYQGGNRTNTGLALRYLS;)HSFLVSQG

H 773 P

---------V-A--HS-R------EV------F------------Q---E---SA---

. . I I DREQAPNLVYMVTGNPASDEIKRLPGDIQWPIGVGPNANVQELERIGWPNAPILIQDF ---E-------------------M---------------DVEM-----LS------F----

H 033 P

.

EtgJ. Alignment of the protein sequence of human vWF with that predicted for porcine vWF. Dashes refer to identical amino acids in these proteins. Gaps (‘) are introduced in the porcine sequence (P) to maintain optimal alignment with human (H). Dots (m) denote conserved amino acids. Members of the following groups of amino acids were considered to be conserved: (M,I,L,V); (F,Y,W); (A,G); (S,T); (QN); (K.R); and (E,D). The solid line above the sequence indicates putative domains that interact with GP lb. Dotted lines refer to botrocetin binding sites. The triangles (A) point out Cys-509 and-695 which form a disulfide loop. The double line (II) indicates the proposed proteolytic cleavage site at Tyr642/Met-643.

sequence

also

residues

suggested

important

differences

with

human

vWF

in these

areas

(18).

Four

contiguous

(478 to 481) found within the first GP lb binding domain of human vWF are absent in the

porcine sequence

and in the second domain, porcine vWF contains two arginine

while there are no positively These differences

charged

amino acids within these segments

between the two species demonstrate

different native conformation from Leu480/Val481

residues (706/707)

in the human sequence.

an area that may contribute to the presumably

of porcine and human vWF. Nevertheless,

a vWF fragment that extends

to Gly 718 contains the binding sites to GP lb suggesting that the sequence Cys-

474 to Gly-479 is not critical for GP lb binding (19). This would include two of the amino acids absent in the pig sequence. only modulate containing

Recently, it has been suggested that the two domains (474 to 488 and 694 to 708)

the ristocetin

proline-rich

mediated

binding of vWF to GP lb (20) because

repeats also inhibited the ristocetin dependent

These areas in the porcine protein maintain glycosylation for proline

a proline backbone.

interaction

In addition, the potential

sites within and adjacent to these areas were also conserved.

in porcine

linked carbohydrate

vWF

eliminates

at Thr-499.

an O-linked

glycosylation

Modification of carbohydrate

irrelevant

peptides

of vWF and GP lb. O-linked

The substitution of Ser-500

site but is compensated

by a potential

O-

side chains have been shown to affect the

interaction of vWF with its receptor (21). Mismatches Willebrand

in the porcine sequence are at the same position as defects in human type IIB von

disease. Amino acid sequences

may be responsible for the conformational

within the disulfide loop formed by Cys 509 and Cys 695

change that modulates the affinity of vWF for GP lb. This loop

contains distinct binding sites for collagen, heparin, sulfated glycolipids and botrocetin.

565

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GPlb

A

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RESEARCH COMMUNICATIONS

GPlb A 694

A474

AND BIOPHYSICAL

708 “A

RR

76% I -------_-

95%

Homology ---

____-

Homology

-----;;;

_______-

;6g

**= EG I

m-91

II -

8421843

-; 676 L

542

i 92% I .I 639

643

Homology

r____________________________I

552

90%

Homology

627

EiCLe_Schematic comparison of the structure of human and porcine vWF. Amino acids 473 to 891 in the human mature vWF subunit are represented. The arrows indicate amino acid differences between the human and porcine vWF in two domains (residues 474 to 488 and 694 to 708) implicated in the binding of vWF to the platelet receptor GP lb and three putative segments involved in botrocetin

binding to vWF (residues 539 to 553, 569 to 583 and 629 to 643). Dots (a) refer to highly conserved amino acid changes and asterisks (‘) designate missing amino acids in the porcine sequence. The

circled residues in the human sequence denote positions type 116vWD. Percent amino acid homology between the by the dotted lines. The disulfide loop formed by Cys-509 proteolytic cleavage site at Tyr-842/Met-843,

GP

site (Asp-514

lb binding

species.

Adjacent

demonstrated

to Glu-542)

to this domain

with point

mutations

and-695

is indicated

botrocetin

binding

that three distinct segments are involved in botrocetin

as well as a proposed

binding to vWF (residues 539 to

Four of the six point mutations

associated

with type IIB von

has increased affinity for the GP lb platelet receptor (25) resulting in increased aggregation of ristocetin

Interestingly,

three

substitution)

perhaps

of these

analogous

point mutations

as three of four mismatches

to the effect of porcine are in the same position

in the porcine sequence

in the two

site (20) and it has been

disease (vWD) are in the first segment of the botrocetin binding site (22-24).

the presence

in human

has been reported (20) and is 79% homologous

is a proposed

553, 569 to 583 and 629 to 643) (14). Willebrand

associated

two species for selected areas of vWF is given

Type IIB vWF of platelets in

vWF with human platelets. (although

not the same

within this small segment (Fig. 4).

Normal human vWF demonstrates

an Arg-543,

Arg-545 and Trp-550 while the porcine protein has a

histidine at each of these positions.

The other two point mutations reported in type IIB vWD (23,26) are

in the second segment of the botrocetin binding site and this segment is highly conserved

between the

two species (Figs. 3 and 4). In contrast, only 60% of the residues were identical in the third segment. The differences

between

human and porcine vWF within the botrocetin binding site underline

another

area that may cause the different characteristics

of human and porcine vWF especially considering

intriguing

binding

relationship

between

the botrocetin

566

the

site, the defects in type IIB vWD and the

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increased affinity for the human GP lb platelet receptor exhibited by both type IIB and porcine vWF. It is likely, however, that an interaction

of different residues from the various functional

required to maintain the native conformation The area surrounding

of porcine vWF that directly aggregates

a proposed proteolytic

domains

may be

human platelets.

cleavage site is nearly identical. A 40 amino acid

segment from Ser-830 to Pro-869 is 95% conserved (Figs. 3 and 4) and includes a proposed proteolytic cleavage site (Tyr-842/Met-843) vWD (27-30).

Abnormal

and decreased

as well as the majority of point mutations associated with human type IIA

cleavage at this site is thought to result in increased vWF degradation

reactivity with ristocetin,

characteristic

of type IIA vWD (7). The putative

products protease

involved is a calpain; however, in vitro studies, using porcine calpains I and II (31) failed to produce the expected human vWF degradation

products; this may be related to species or tissue dependency

the fact that calpains are intracellular

or membrane

bound (7). The conserved

the proteolytic cleavage site indicates that the protease

sequence

or to

surrounding

involved should have the same specificity in

the two species. The high degree of homology as well as the position of point mutations associated with type IIA vWD suggest that a strict conformation The comparative for a systematic approach molecule further

for mutagenic

permit the design

sequencing

may be required in this area.

analysis of the primary structure of porcine and human vWF provides a rationale studies in both species.

of oligonucleotides

The highly conserved

areas of the

that could be useful for amplification

and direct

of vWF DNA from other species. Due to the species variability in the reaction with ristocetin

comparative

studies on vWF should provide

important

insights

on the complex

adhesive

function of vWF.

Acknowledgments This work was supported in pan by a grant from the Phillippe Foundation, Paris and New York to B. R. Bahnak. V. Ferreira was supported by a pre-doctoral fellowship from Ministere de la Recherche et de la Technologie.

References 1. 2. 3. 4. 5. ;: 8. 9. 10. 11. 12. 13. 14. 15.

Baruch, D., Bahnak, B. Ft., Gina, J.-P., and Meyer, D. (1989) In Bailliere’s Clinical Haematology (J.P. Caen, Ed.), Vol. Ill, pp. 627-672. Bailliere Tindall, London. Sakarfassen, K.S., Bolhuis, P.A., and Sixma, J.J. (1979) Nature. 279, 630-638. Howard. MA.. and Firkin. B.G. (1971) Thromb. Haemost. 26.362-369. Read, M.S., Shermer, R.W., and Brfnkhous, K.M. (1978) . . Pro& Natl. Acad. Sci. USA. 75.4514451 8. Brfnkhous, KM., Thomas, B.D., Ibrahim, S.A., and Read, S. (1977) Thromb. Res. 11, 345-355. Forbes, C.D.. Barr, R.D.. McNicol. G.P.. and Doualas. A..% (1972) J. Clin. Path. 25. 210-217 Dent, JlA., Berkowhz, S.-D., Ware; J., K&per, C.t?., and Ruggert, Z.M. (1990) Proc.‘Natl. Acad. Sci. USA. 87, 6306-6310. Wu, Q.Y., Bahnak, B.R., Coulombel, L., Kerbiriou-Nabias, D., Drouet, L., Pietu, G., Meulien, P., Pavirani, A., Caen, J.P., and Meyer, D. (1988) Blood. 71, 1341-1346. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B., and Erlich, H.A. (1988) Science. 239, 487-491. Casanova J.-L., Pannetier, C., Jaulin, C.,and Kourilsky, P. (1990) Nucleic Acids Res. 18,4028. Bonthron, D., Orr, EC., Mitsock, L.M., Ginsburg, D., Handin, R.I., and Orkin, S.H. (1986) Nucleic Acids Res. 14, 7125-7127. Shelton-lnloes, B.B., Titani, K., and Sadler, J.E. (1986) Biochemistry. 25,3164-3171. Christophe, O., Obert, B., Meyer, D., and Girma, J.-P. (1991) Blood 78, (in press). Sugimoto, M., Mohri, H., McClintock, R.A., and Ruggerf, Z.M. (1991) J. Biol. Chem 266, 1817218178. Andrew% R.K., Booth, W.J., Gorman, J.J., Castaldi, P.A., and Berndt, MC. (1989) Biochemistry. 28, 8317-8326. 567

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