1. PAR3 AS A LINK BETWEEN BLOODPAR3 AS A LINK BETWEEN BLOOD
COAGULATION AND IMMUNITY:COAGULATION AND IMMUNITY:
EXPRESSION AND FUNCTION INEXPRESSION AND FUNCTION IN
LYMPHOCYTESLYMPHOCYTES
Petrova Yu.I.,Petrova Yu.I., Kameneva O.V., Zhukova A.I.,Kameneva O.V., Zhukova A.I.,
Scudder L.*, Bahou W.F. * and Skok M.V.Scudder L.*, Bahou W.F. * and Skok M.V.
Palladin Institute of Biochemistry of the NAS of Ukraine, Kyiv,Palladin Institute of Biochemistry of the NAS of Ukraine, Kyiv,
UkraineUkraine
* State University of New York at Stony Brook, Stony Brook, NY,* State University of New York at Stony Brook, Stony Brook, NY,
USAUSA

2. Cellular effects of ThrombinCellular effects of Thrombin
Shaun R. Coughlin. Nature, Vol. 407, 14 September 2000, pp. 258-264

3. Protease-activated receptors (PARs) are
members of Rhodopsin-like GPCR subfamily
For the moment 4
different members
are discovered:
PAR1
PAR2
PAR3
PAR4
Lawrence F. Brass. Chest,
2003; 124, pp. 18S–25S

4. VS Ossovskaya & NW Bunnett. Physiol Rev 84: 579–621, 2004
PAR tissue distribution and functions

5. TheThe aimaim of the work was to studyof the work was to study
PAR3 expression in mouse bloodPAR3 expression in mouse blood
related cells and their function inrelated cells and their function in
lymphocytes usinglymphocytes using
cleavage site specific monoclonalcleavage site specific monoclonal
antibodies 8E8.antibodies 8E8.

6. Characterization of mAbCharacterization of mAb
8E8:8E8:
((AA)) -- sequences of antigenicsequences of antigenic
human and corresponding mousehuman and corresponding mouse
PAR3 peptides;PAR3 peptides;
((BB) - binding of affinity purified) - binding of affinity purified
mAb 8E8 to hPAR3 (31-47);mAb 8E8 to hPAR3 (31-47);
hPAR1 (30-99); keyhole limpethPAR1 (30-99); keyhole limpet
hemocyanin (KLH) and bovinehemocyanin (KLH) and bovine
serum albumin (BSA) studied byserum albumin (BSA) studied by
ELISA;ELISA;
((CC) - Western blot of HEp-2 cell) - Western blot of HEp-2 cell
lysates developed by mAb 8E8: 1 –lysates developed by mAb 8E8: 1 –
non-transfected cells; 2 – PAR3-non-transfected cells; 2 – PAR3-
transfected cells; 3 – PAR3 anti-transfected cells; 3 – PAR3 anti-
sense-transfected cells.sense-transfected cells.
AKPTLPIKTFRGAPPNS (PAR3, human, 31-47)
AKPTLTIKSFNGGPQNT (PAR3, mouse, 30-46)
A
B
C

7. A B
Aggregation,%
C
IgG2b
mAb
8E8
Interaction of mAb 8E8 with purified mouse platelets: flow
cytometry dot-plots of platelets stained with non-specific IgG2b
(A) or mAb 8E8 (B) and the curves of platelet aggregation
induced by 3 nM thrombin in the presence of either non-specific
IgG2b or mAb 8E8 (C).

8. A B
C
D
CD45R (B220)mAb8E8CD45R (B220)
mouseIgG2b
PA
Flow cytometry dot-plots
of mouse splenocytes
double-stained with either
non-specific IgG2b (A) or
mAb 8E8 (B) and anti-
B220 mAb.
C – The part of PAR3+
cells in different
populations of mouse
spleen cells, according to
flow cytometry data. *
- P<0.05, *** - P<0.005,
NS – non-significant
compared to the binding
of non-specific IgG2b.
D – Western blot of
magnetically sorted
mouse T (1) and B (2)
lymphocytes
immunoprecipitated and
developed with mAb 8E8.
PAR3 presence in mouse blood-
related cells

9. MAb 8E8 binding to hybridoma 1D6 in flow
cytometry. Thrombin was added to the cells 15 min prior to the mAb. Either BSA
or PAR3 (31-47)-BSA were pre-incubated with the mAb overnight (6.5 μg per 1 μg mAb).
** - p<0.005 compared to mAb 8E8 alone (for thrombin) or to BSA (for PAR3-BSA).

10. A B
Proliferation of hybridoma 1D6: A, in the presence of thrombin
and/or PAR4 (right and inverted) and PAR3 peptides; B, in the presence of mAbs 2E3
and 8E8 (20 μg/ml,). ** - p<0.005; *** - p<0.0005 compared to the non-treated culture
(the first column).

11. [3H]-Thymidineincorporation,%
The effect of thrombin and/or mAb 8E8 (20 μg/ml) on B (anti-
CD40) and T (anti-CD3) lymphocyte activation in the total
splenocyte culture. The thrombin activity of 0.1, 1 and 10 NIH U/ml (T0.1,
T1 and T10, respectively) approximately corresponds to 1, 10 and 100 nM of
thrombin. *** - P<0.005, NS – non-significant compared to control value.

12. IntracellularIntracellular Са2+Са2+ level in Fura-2 loaded mouse plateletslevel in Fura-2 loaded mouse platelets
and splenocytes in the presence of 8E8 mAb or non-and splenocytes in the presence of 8E8 mAb or non-
specific IgG2b.specific IgG2b.
8E8m Ab
untreated
IgG2b
platelets
splenocytes

13. 2D electrophoresis of immunoprecipitated cell2D electrophoresis of immunoprecipitated cell
lysate of hybridoma 1D6 (A) and whole celllysate of hybridoma 1D6 (A) and whole cell
lysates of resting (B) and CD3/CD40-activated (C)lysates of resting (B) and CD3/CD40-activated (C)
mouse splenic cellsmouse splenic cells
A
B
C

14. Conclusions:Conclusions:
Mouse lymphocytes and myeloid cellsMouse lymphocytes and myeloid cells
express PAR3 on protein level. B lymphocytesexpress PAR3 on protein level. B lymphocytes
demonstrate the highest level of expression;demonstrate the highest level of expression;
PAR3 receptor on B lymphocytes isPAR3 receptor on B lymphocytes is
functionally active and involved in thefunctionally active and involved in the
regulation of lymphocyteregulation of lymphocyte
proliferation/activation;proliferation/activation;
Receptors of PAR family comprise theReceptors of PAR family comprise the
important link between immunity and bloodimportant link between immunity and blood
coagulation.coagulation.

15. Acknowledgements. We are grateful to Drs. T. M. Platonova and
O. M. Savchuk for thrombin activity testing and mAb purification; to Drs. S.
Sochilnytskiy and G. Dubrovska for help with 2D electrophoresis.
The work was supported with Civilian Research and
Development Foundation (CRDF) grant UB1-2433-KV-02 and
FEBS Short-Term Fellowship (Yu. Petrova)