Presentation-Moscow2005

PAR3 AS A LINK BETWEEN BLOOD PAR3 AS A LINK BETWEEN BLOOD COAGULATION AND IMMUNITY: COAGULATION AND IMMUNITY:

EXPRESSION AND FUNCTION IN EXPRESSION 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

Cellular effects of ThrombinCellular effects of Thrombin

Shaun R. Coughlin. Nature, Vol. 407, 14 September 2000, pp. 258-264

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

VS Ossovskaya & NW Bunnett. Physiol Rev 84: 579–621, 2004

PAR tissue distribution and functions

The The aimaim of the work was to study of the work was to study PAR3 expression in mouse blood PAR3 expression in mouse blood related cells and their function in related cells and their function in

lymphocytes using lymphocytes using

cleavage site specific monoclonal cleavage site specific monoclonal antibodies 8E8. antibodies 8E8.

Characterization of mAb Characterization of mAb 8E8:8E8:

((AA)) -- sequences of antigenic sequences of antigenic human and corresponding mouse human 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 limpet hPAR1 (30-99); keyhole limpet hemocyanin (KLH) and bovine hemocyanin (KLH) and bovine

serum albumin (BSA) studied by serum 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

A B

Ag

gre

gat

ion

, %

CIgG2b

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).

A B

C

D

CD45R (B220)m

Ab

8E

8CD45R (B220)

mo

us

e Ig

G2b

PA

R3-

posi

tive

cells

, %

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

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).

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).

[3H

]-T

hym

idin

e in

corp

ora

tio

n,

%

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.

Intracellular Intracellular Са2+ Са2+ level in Fura-2 loaded mouse platelets level 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

untreatedIgG2b

platelets

splenocytes

2D electrophoresis of immunoprecipitated cell 2D electrophoresis of immunoprecipitated cell lysate of hybridoma 1D6 (A) and whole cell lysate 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

Conclusions:Conclusions:

Mouse lymphocytes and myeloid cells Mouse lymphocytes and myeloid cells express PAR3 on protein level. B lymphocytes express PAR3 on protein level. B lymphocytes demonstrate the highest level of expression; demonstrate the highest level of expression;

PAR3 receptor on B lymphocytes is PAR3 receptor on B lymphocytes is functionally active and involved in the functionally active and involved in the regulation of lymphocyte regulation of lymphocyte proliferation/activation;proliferation/activation;

Receptors of PAR family comprise the Receptors of PAR family comprise the important link between immunity and blood important link between immunity and blood coagulation.coagulation.

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)