Cell-based hemostasis vs. platelet-based hemostasis

http://www.akh-wien.ac.at/biomed-research/htx/platweb1.htm

http://www.akh-wien.ac.at/biomed-research/htx/platweb1.htm

Asimetría de fosfolípidos de la membrana celularActividad procoagulante de las plaquetas

EscramblasaEscramblasa TranslocasaTranslocasaFlopasaFlopasa

ProtrombinaProtrombina

TrombinaTrombina

MacrófagoMacrófago

VesículaVesícula

CalpaínaCalpaína

CitoesqueletCitoesqueletoo

Hemophilia

Coagulation

Figure 1

A

200 bp

600 bp

B

200 bp

600 bp

200 bp

600 bp

C

2000 bp

1500 bp

2000 bp

CD14β-acti

n

TF GPIbβ-a

ctin

CD14TF GPIb

Non-stimulated PBMC

PBMC +TRAP

Non-stimulated platelets

TRAP-stimulated platelets

Non-stimulated PBMC

LPS-stimulated PBMC

β-actin

CD14TF GPIb

β-actin

CD14TF GPIb

β-actin

CD14TF GPIb

β-actin

TF

1 2 3 4 5

47 kDa

A

IgG Basal 30 60 90 min min min

B

47 kDa

37 kDa

64.2 kDa

83 kDa

1 2 3 4

Basal 30 60 90 min min min

1 2 3 4 5

47 kDa

A

IgG Basal 30 60 90 min min min

B

47 kDa

37 kDa

64.2 kDa

83 kDa

1 2 3 4

Basal 30 60 90 min min min

Figure 2

1 2 3 4 5

47 kDa

A

IgG Basal 30 60 90 min min min

B

47 kDa

37 kDa

64.2 kDa

83 kDa

1 2 3 4

Basal 30 60 90 min min min

A

60.4 kDa

47.2 kDa

35 kDa

TRAP – – + +Puromycin – + – +

Figure 3-S

7,5 mPearson Coeff. : 0.1855

Pearson Coeff. : 0.3188

Figure 3

7,5 m

TF GPIb Merge

62.0 kDa

47.5 kDa

32.5 kDa

1 2 3 4

TRAP – – + +Puromycin – + – +

1 2 3 4 5 6 7 8

TRAP – + – + – + – +Tunicamycin – – + + – – + +Puromycin – – – – + + – –

LPS – + + – + +Puromycin – + – – – +

109. 5 kDa

78.9 kDa

60.4 kDa

47.2 kDa

35.1 kDa

24.9 kDa

109.5 kDa

1 2 3 4 5 6

78.9 kDa

60.4 kDa

47.2 kDa

35.1 kDa

24.9 kDa

Figure 4

*

A. Platelets B. Platelets

C. PBMC suspensions

62.0 kDa

47.5 kDa

32.5 kDa

1 2 3 4

TRAP – – + +Puromycin – + – +

1 2 3 4 5 6 7 8

TRAP – + – + – + – +Tunicamycin – – + + – – + +Puromycin – – – – + + – –

LPS – + + – + +Puromycin – + – – – +

109. 5 kDa

78.9 kDa

60.4 kDa

47.2 kDa

35.1 kDa

24.9 kDa

109.5 kDa

1 2 3 4 5 6

78.9 kDa

60.4 kDa

47.2 kDa

35.1 kDa

24.9 kDa

Figure 4

B. Platelets

*

1 2 3 4

62.0 kDa

47.5 kDa32.5 kDa

LPS + + + + Actinomycin D – + – – DRB – – + –Puromycin – – – +

Figure 4S

MergeGPIbαTF

3 m

Hoechst Vis

Figure 5

Merge

GPIb

GPIb

TF

TF

VisTF GPIbα

TF GPIbα Merge

Figure 5-S

TFGPIb

Merge

GPIbα TF

Merge

GPIbα TF

Merge

Figure 6

GPIbα TF

Merge

6 μM

0 25 50 75 100 1250

10

20

30 DMSO

Puromycin

Time (minutes)

Pro

co

ag

ula

nt

ac

tiv

ity

(AU

/ 2

x1

07 p

late

lets

)

0 25 50 75 100 1250

10

20

30 DMSO

Puromycin

Time (minutes)

Pro

co

ag

ula

nt

ac

tiv

ity

(AU

/ 2

x1

07 p

late

lets

)

Figure 1-S

0 15 60 1200

50

100

150

200

250

Control

aPC

CTI

-FVIII:C

-TF

-FVIIa

Time (min)Activation with 5M TRAP

Pro

coag

ula

nt

Act

ivit

y (%

)

Figure 2-S

Regular ManuscriptHuman platelets synthesize and express functional tissue factor.

Olga Panes1, Valeria Matus1, Claudia G. Sáez1, Teresa Quiroga2, Jaime Pereira1, Diego Mezzano1.

From the Departments of Hematology-Oncology1, and Clinical Laboratory2, School of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile.

Supported by Grants 8010002 (D.M., J.P., T.Q.) and 1060637 (D.M.) from Fondecyt, Chile.

(Blood, 2007, en prensa)