Key Points

  • PSplt controls platelet activation as well as coagulation in thrombi in large veins, but not in large arteries.

  • PSplt limits thrombin generation in the thrombus and ensures that highly activated platelets and fibrin remain localized at the injury site.

Abstract

Anticoagulant protein S (PS) in platelets (PSplt) resembles plasma PS and is released on platelet activation, but its role in thrombosis has not been elucidated. Here we report that inactivation of PSplt expression using the Platelet factor 4 (Pf4)-Cre transgene (Pros1lox/loxPf4-Cre+) in mice promotes thrombus propensity in the vena cava, where shear rates are low, but not in the carotid artery, where shear rates are high. At a low shear rate, PSplt functions as a cofactor for both activated protein C and tissue factor pathway inhibitor, thereby limiting factor X activation and thrombin generation within the growing thrombus and ensuring that highly activated platelets and fibrin remain localized at the injury site. In the presence of high thrombin concentrations, clots from Pros1lox/loxPf4-Cre mice contract, but not clots from Pros1lox/loxPf4-Cre+ mice, because of highly dense fibrin networks. Thus, PSplt controls platelet activation as well as coagulation in thrombi in large veins, but not in large arteries.

REFERENCES

REFERENCES
1.
Di Scipio
RG
,
Hermodson
MA
,
Yates
SG
,
Davie
EW
.
A comparison of human prothrombin, factor IX (Christmas factor), factor X (Stuart factor), and protein S
.
Biochemistry
.
1977
;
16
(
4
):
698
-
706
.
2.
Griffin
JH
,
Zlokovic
BV
,
Mosnier
LO
.
Protein C anticoagulant and cytoprotective pathways
.
Int J Hematol
.
2012
;
95
(
4
):
333
-
345
.
3.
Wildhagen
KC
,
Lutgens
E
,
Loubele
ST
,
ten Cate
H
,
Nicolaes
GA
.
The structure-function relationship of activated protein C. Lessons from natural and engineered mutations
.
Thromb Haemost
.
2011
;
106
(
6
):
1034
-
1045
.
4.
Rezaie
AR
.
Regulation of the protein C anticoagulant and antiinflammatory pathways
.
Curr Med Chem
.
2010
;
17
(
19
):
2059
-
2069
.
5.
Dahlbäck
B
,
Villoutreix
BO
.
The anticoagulant protein C pathway
.
FEBS Lett
.
2005
;
579
(
15
):
3310
-
3316
.
6.
Dahlbäck
B
.
Pro- and anticoagulant properties of factor V in pathogenesis of thrombosis and bleeding disorders
.
Int J Lab Hematol
.
2016
;
38
(
Suppl 1
):
4
-
11
.
7.
Hackeng
TM
,
Seré
KM
,
Tans
G
,
Rosing
J
.
Protein S stimulates inhibition of the tissue factor pathway by tissue factor pathway inhibitor
.
Proc Natl Acad Sci USA
.
2006
;
103
(
9
):
3106
-
3111
.
8.
Chattopadhyay
R
,
Sengupta
T
,
Majumder
R
.
Inhibition of intrinsic Xase by protein S: a novel regulatory role of protein S independent of activated protein C
.
Arterioscler Thromb Vasc Biol
.
2012
;
32
(
10
):
2387
-
2393
.
9.
Mahasandana
C
,
Suvatte
V
,
Marlar
RA
,
Manco-Johnson
MJ
,
Jacobson
LJ
,
Hathaway
WE
.
Neonatal purpura fulminans associated with homozygous protein S deficiency
.
Lancet
.
1990
;
335
(
8680
):
61
-
62
.
10.
Comp
PC
,
Esmon
CT
.
Recurrent venous thromboembolism in patients with a partial deficiency of protein S
.
N Engl J Med
.
1984
;
311
(
24
):
1525
-
1528
.
11.
Schwarz
HP
,
Fischer
M
,
Hopmeier
P
,
Batard
MA
,
Griffin
JH
.
Plasma protein S deficiency in familial thrombotic disease
.
Blood
.
1984
;
64
(
6
):
1297
-
1300
.
12.
Castoldi
E
,
Maurissen
LF
,
Tormene
D
, et al
.
Similar hypercoagulable state and thrombosis risk in type I and type III protein S-deficient individuals from families with mixed type I/III protein S deficiency
.
Haematologica
.
2010
;
95
(
9
):
1563
-
1571
.
13.
Makris
M
,
Leach
M
,
Beauchamp
NJ
, et al
.
Genetic analysis, phenotypic diagnosis, and risk of venous thrombosis in families with inherited deficiencies of protein S
.
Blood
.
2000
;
95
(
6
):
1935
-
1941
.
14.
Ten Kate
MK
,
Platteel
M
,
Mulder
R
, et al
.
PROS1 analysis in 87 pedigrees with hereditary protein S deficiency demonstrates striking genotype-phenotype associations
.
Hum Mutat
.
2008
;
29
(
7
):
939
-
947
.
15.
Lijfering
WM
,
Mulder
R
,
ten Kate
MK
,
Veeger
NJ
,
Mulder
AB
,
van der Meer
J
.
Clinical relevance of decreased free protein S levels: results from a retrospective family cohort study involving 1143 relatives
.
Blood
.
2009
;
113
(
6
):
1225
-
1230
.
16.
Vossen
CY
,
Conard
J
,
Fontcuberta
J
, et al
.
Risk of a first venous thrombotic event in carriers of a familial thrombophilic defect. The European Prospective Cohort on Thrombophilia (EPCOT)
.
J Thromb Haemost
.
2005
;
3
(
3
):
459
-
464
.
17.
Roach
RE
,
Lijfering
WM
,
van Hylckama Vlieg
A
,
Helmerhorst
FM
,
Rosendaal
FR
,
Cannegieter
SC
.
The risk of venous thrombosis in individuals with a history of superficial vein thrombosis and acquired venous thrombotic risk factors
.
Blood
.
2013
;
122
(
26
):
4264
-
4269
.
18.
Vossen
CY
,
Walker
ID
,
Svensson
P
, et al
.
Recurrence rate after a first venous thrombosis in patients with familial thrombophilia
.
Arterioscler Thromb Vasc Biol
.
2005
;
25
(
9
):
1992
-
1997
.
19.
Lijfering
WM
,
Brouwer
JL
,
Veeger
NJ
, et al
.
Selective testing for thrombophilia in patients with first venous thrombosis: results from a retrospective family cohort study on absolute thrombotic risk for currently known thrombophilic defects in 2479 relatives
.
Blood
.
2009
;
113
(
21
):
5314
-
5322
.
20.
Pintao
MC
,
Ribeiro
DD
,
Bezemer
ID
, et al
.
Protein S levels and the risk of venous thrombosis: results from the MEGA case-control study
.
Blood
.
2013
;
122
(
18
):
3210
-
3219
.
21.
Koster
T
,
Rosendaal
FR
,
Briët
E
, et al
.
Protein C deficiency in a controlled series of unselected outpatients: an infrequent but clear risk factor for venous thrombosis (Leiden Thrombophilia Study)
.
Blood
.
1995
;
85
(
10
):
2756
-
2761
.
22.
Ogura
M
,
Tanabe
N
,
Nishioka
J
,
Suzuki
K
,
Saito
H
.
Biosynthesis and secretion of functional protein S by a human megakaryoblastic cell line (MEG-01)
.
Blood
.
1987
;
70
(
1
):
301
-
306
.
23.
Schwarz
HP
,
Heeb
MJ
,
Wencel-Drake
JD
,
Griffin
JH
.
Identification and quantitation of protein S in human platelets
.
Blood
.
1985
;
66
(
6
):
1452
-
1455
.
24.
Camire
RM
,
Pollak
ES
,
Kaushansky
K
,
Tracy
PB
.
Secretable human platelet-derived factor V originates from the plasma pool
.
Blood
.
1998
;
92
(
9
):
3035
-
3041
.
25.
Saller
F
,
Brisset
AC
,
Tchaikovski
SN
, et al
.
Generation and phenotypic analysis of protein S-deficient mice
.
Blood
.
2009
;
114
(
11
):
2307
-
2314
.
26.
Prince
R
,
Bologna
L
,
Manetti
M
, et al
.
Targeting anticoagulant protein S to improve hemostasis in hemophilia
.
Blood
.
2018
;
131
(
12
):
1360
-
1371
.
27.
Hisada
Y
,
Yasunaga
M
,
Hanaoka
S
, et al
.
Discovery of an uncovered region in fibrin clots and its clinical significance
.
Sci Rep
.
2013
;
3
(
1
):
2604
.
28.
Aghourian
MN
,
Lemarié
CA
,
Blostein
MD
.
In vivo monitoring of venous thrombosis in mice
.
J Thromb Haemost
.
2012
;
10
(
3
):
447
-
452
.
29.
Novotny
J
,
Chandraratne
S
,
Weinberger
T
, et al
.
Histological comparison of arterial thrombi in mice and men and the influence of Cl-amidine on thrombus formation
.
PLoS One
.
2018
;
13
(
1
):
e0190728
.
30.
Tiedt
R
,
Schomber
T
,
Hao-Shen
H
,
Skoda
RC
.
Pf4-Cre transgenic mice allow the generation of lineage-restricted gene knockouts for studying megakaryocyte and platelet function in vivo
.
Blood
.
2007
;
109
(
4
):
1503
-
1506
.
31.
Burstyn-Cohen
T
,
Heeb
MJ
,
Lemke
G
.
Lack of protein S in mice causes embryonic lethal coagulopathy and vascular dysgenesis
.
J Clin Invest
.
2009
;
119
(
10
):
2942
-
2953
.
32.
Schwarz
HP
,
Heeb
MJ
,
Lämmle
B
,
Berrettini
M
,
Griffin
JH
.
Quantitative immunoblotting of plasma and platelet protein S
.
Thromb Haemost
.
1986
;
56
(
3
):
382
-
386
.
33.
Griffin
JH
,
Gruber
A
,
Fernández
JA
.
Reevaluation of total, free, and bound protein S and C4b-binding protein levels in plasma anticoagulated with citrate or hirudin
.
Blood
.
1992
;
79
(
12
):
3203
-
3211
.
34.
Dahlbäck
B
.
The tale of protein S and C4b-binding protein, a story of affection
.
Thromb Haemost
.
2007
;
98
(
1
):
90
-
96
.
35.
Fernández
JA
,
Heeb
MJ
,
Xu
X
,
Singh
I
,
Zlokovic
BV
,
Griffin
JH
.
Species-specific anticoagulant and mitogenic activities of murine protein S
.
Haematologica
.
2009
;
94
(
12
):
1721
-
1731
.
36.
Angelillo-Scherrer
A
,
Fontana
P
,
Burnier
L
, et al
.
Connexin 37 limits thrombus propensity by downregulating platelet reactivity
.
Circulation
.
2011
;
124
(
8
):
930
-
939
.
37.
Angelillo-Scherrer
A
,
de Frutos
P
,
Aparicio
C
, et al
.
Deficiency or inhibition of Gas6 causes platelet dysfunction and protects mice against thrombosis
.
Nat Med
.
2001
;
7
(
2
):
215
-
221
.
38.
Angelillo-Scherrer
A
,
Burnier
L
,
Flores
N
, et al
.
Role of Gas6 receptors in platelet signaling during thrombus stabilization and implications for antithrombotic therapy
.
J Clin Invest
.
2005
;
115
(
2
):
237
-
246
.
39.
Solomon
C
,
Ranucci
M
,
Hochleitner
G
,
Schöchl
H
,
Schlimp
CJ
.
Assessing the methodology for calculating platelet contribution to clot strength (platelet component) in thromboelastometry and thrombelastography
.
Anesth Analg
.
2015
;
121
(
4
):
868
-
878
.
40.
Weiss
EJ
,
Hamilton
JR
,
Lease
KE
,
Coughlin
SR
.
Protection against thrombosis in mice lacking PAR3
.
Blood
.
2002
;
100
(
9
):
3240
-
3244
.
41.
Macrae
FL
,
Duval
C
,
Papareddy
P
, et al
.
A fibrin biofilm covers blood clots and protects from microbial invasion
.
J Clin Invest
.
2018
;
128
(
8
):
3356
-
3368
.
42.
Lam
WA
,
Chaudhuri
O
,
Crow
A
, et al
.
Mechanics and contraction dynamics of single platelets and implications for clot stiffening
.
Nat Mater
.
2011
;
10
(
1
):
61
-
66
.
43.
Harmon
S
,
Preston
RJ
,
Ni Ainle
F
, et al
.
Dissociation of activated protein C functions by elimination of protein S cofactor enhancement
.
J Biol Chem
.
2008
;
283
(
45
):
30531
-
30539
.
44.
Winckers
K
,
Thomassen
S
,
Ten Cate
H
,
Hackeng
TM
.
Platelet full length TFPI-α in healthy volunteers is not affected by sex or hormonal use
.
PLoS One
.
2017
;
12
(
2
):
e0168273
.
45.
Maurissen
LF
,
Castoldi
E
,
Simioni
P
,
Rosing
J
,
Hackeng
TM
.
Thrombin generation-based assays to measure the activity of the TFPI-protein S pathway in plasma from normal and protein S-deficient individuals
.
J Thromb Haemost
.
2010
;
8
(
4
):
750
-
758
.
46.
Brass
LF
,
Zhu
L
,
Stalker
TJ
.
Minding the gaps to promote thrombus growth and stability
.
J Clin Invest
.
2005
;
115
(
12
):
3385
-
3392
.
47.
Tomaiuolo
M
,
Matzko
CN
,
Poventud-Fuentes
I
,
Weisel
JW
,
Brass
LF
,
Stalker
TJ
.
Interrelationships between structure and function during the hemostatic response to injury
.
Proc Natl Acad Sci USA
.
2019
;
116
(
6
):
2243
-
2252
.
48.
Welsh
JD
,
Poventud-Fuentes
I
,
Sampietro
S
,
Diamond
SL
,
Stalker
TJ
,
Brass
LF
.
Hierarchical organization of the hemostatic response to penetrating injuries in the mouse macrovasculature
.
J Thromb Haemost
.
2017
;
15
(
3
):
526
-
537
.
49.
Stalker
TJ
,
Welsh
JD
,
Tomaiuolo
M
, et al
.
A systems approach to hemostasis: 3. Thrombus consolidation regulates intrathrombus solute transport and local thrombin activity
.
Blood
.
2014
;
124
(
11
):
1824
-
1831
.
50.
Radu
CM
.
Study of the origin of platelets coagulation protein S by human megakaryocyte cultures and characterization of platelets protein S in patients with inherited protein S deficiency [thesis]
.
Padua
:
University of Padua
;
2009
.
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