Bo Nilsson, Uppsala University, Sweden

Thromboinflammation in therapeutic medicine

Höstmöte KITM 30-31th of August, 2017, Uppsala

Sowa et al, J Biomed Opt, 2017

Thromboinflammation

Clot

Inflammation

Proliferation

Remodelling

Markiewski, Trends Immunol, 2007

The cascade systems of the blood

Coagulation

Complement

C3 deficiency leads to prolonged bleeding time

Bauer et al., PlosOne, 2011

Gushiken et al., J Thromb Haemost, 2009

C3 KO mice have attenuated plateletaggregation and prolonged bleeding time

Darbousset R, Blood, 2012; 2014.

Formation of PMN (CD16+)-platelet (CD42a+) complexes in whole blood from a C3 deficient

person

Hamad, Thrombosis Haemostasis, 2015

CD16/CD42a complexesCD11b upregulation

Thrombo-inflammation

Tissue Factor

Lectin pathway

Classical pathway

Alternative pathway C3a, C5a

MBL

Ficolins

Properdin

C1q, CRP

Contact system

Tissue factor pathway

Thrombin

TF-FVII

FXII

platelets

Fibrin Fibrin

platelets

Thrombin

IschemiaCell stress

Immune complexes

Thromboinflammation triggered by

complement

Platelet

MAC

ComplementdysregulationImmune

complexes

C3aC5a

Endothelium

C3aC5aMAC

C3aC5a

1. Complement activation triggered by C1q

Hamad et al., JTH, 2009

Binding of complement components to

activated platelets

fH

Non Act. Act.0

50

100

150 **

C4BP

Non Act. Act.0

20

40

60 *

C1INH

Non Act. Act.0

25

50

75

100*

CS

Non Act. Act.0

100

200

300

400

Hamad et a., J Immunol, 2010

Blocking Complement Activation

Blocking at the C1q and C3

levels or activation in the

presence of EDTA, EGTA did

not affect the binding.

0

250

500

750

* *

**

n.s.

Hamad, J Immunol, 2010

Platelet

TRAP

PAR-1, 4C3H2O

2. C3H2O acts as a ligand for leukocyte

receptors CR1 and CR3

C3H2O acts as a ligand for:

1. CR1 (CD35)

2. CR3 (CD11b/CD18)

on PMNs and monocytes

Summary

The specificity of MASP-1 and -2

Generation of MASP-AT/C1INH Complexes by

TRAP activated platelets

MASP1/3 - AT

SERU

M

EDTA

PLA

SMA

Hiru

din P

PPPR

P 0 m

in

PRP 1

5 m

in

PRP 3

0 m

in

PRP 6

0 m

in

0

1

2

3

4

Me

an

of O

D v

alu

e

ns**

***

MASP1/3 - C1INH

Me

an

of O

D v

alu

e

SERU

M

EDTA

PLA

SMA

Hiru

din P

PPPR

P 0 m

in

PRP 1

5 m

in

PRP 3

0 m

in

PRP 6

0 m

in0

1

2

3

4ns

**

ns

MASP2 - AT

SERU

M

EDTA

PLA

SMA

Hiru

din P

PPPR

P 0 m

in

PRP 1

5 m

in

PRP 3

0 m

in

PRP 6

0 m

in

0

1

2

3

4

Me

an

of O

D v

alu

e

*

**

**

MASP2 - C1INH

Me

an o

f OD

va

lue

SERU

M

EDTA

PLA

SMA

Hiru

din P

PPPR

P 0 m

in

PRP 1

5 m

in

PRP 3

0 m

in

PRP 6

0 m

in

0

1

2

3

ns

**

Kozarcanin et al., JTH, 2016

MASP-1,-2/serpin complexes

Koz

Activation of PPP by glas

Kozarcanin et al., JTH, 2016

3. Platelet- and fibrin-mediated lectin

pathway activation

Platelet

TRAP, thrombin

PAR-1, 4

LP activation

Kozarcanin et al., JTH, 2016

1. DIC

2. Thrombotic events such as cardiac infarction, stroke and other

cardiovascular conditions

3. Biomaterials implants (joint replacements, scaffolds for tissue

engineering etc), extracorporeal treatments (hemodialysis,

cardiopulmonary bypass)

4. Pharmacological delivery systems e.g. lipid micelles, polymers, virus

vectors etc.

5. Microangiopathies- dysregulation (aHUS)

6. Cell and cell cluster transplantation and therapies.

7. Whole organ transplantation

1. Rheumatic disease (scleroderma, SLE, antiphospholipid syndrome).

Thromboinflammation is an important

pathophysiological mechanism in several clinical

conditions and treatments

1. Thromboinflammation in

hemodialysis

Ekdahl et al., Nature Rev Neph, 2017

2. Thromboinflammation in transplantation

Characterized by:

• Coagulation and complement

activation

• Platelet consumption

• Leukocyte activation and infiltration

• Cytokine generation

• Cell death

• Graft loss / delayed graft function

Mechanism:

• Antibodies and lectins• Lack of cell associated regulators

e.g. DAF, MCP, AT, C1-INH• Cell stress- cyto/chemokines, TF

expression

I/R injury

IBMIR

Ischemia/reperfusion injury

Conjugation of peptides or macromolecules phospholipid

PEG-phospholipid linker

-Cys-

PEG

Engberg, Biomaterials, 2009, Nilsson Biomaterials, 2012; Asif, Acta Biomaterialia, 2015

Hydrophobic

interaction

Short peptide-

PEG-lipid

Heparin conjugates

Cell membrane

0

200

400

600

800

1000

1200

0 20 40 60

An

gle

sh

ift

(mD

A)

Time (min)

control peptide

HBPIII

CHC AT

Asif, Acta Biomaterialia, 2015

Heparin-coating

Inflammation, cytolysis (C5a, C3a)

Recruited factor H

Immobilized apyrase(CD39)

A. Regulation of the complement system

Platelet aggregation

B. Regulation of platelet activation (and coagulation system)

Regulation of the complement and coagulation

system by autoregulation

Teramura, Biomaterials, 2013

Slide chamber model experiment

Microscope slide glass

Porcine aortic endothelial cells

Factor H on PAEC after exposure with whole blood

5% fH-bp-PEG-

lipid/PAEC Control PAEC

Nilsson, Biomaterials, 2013

Whole blood experiment on

Apyrase/5C6-immobilized surface

(a) (b)

(c) (d)

Protection of transplanted kidneys against I/R in two models

Donors

n=6

Recipient A, not

related to donor

(n=6)

Recipient B, not

related to donor

(n=6)

Long-term, allogeneic

Donors

N=7

Recipient, not

related to donor

(n=7)

En bloc, short-term

Binding of PEG-phospholipid

*

50 100 150 200 250 300 350 400 450 500 550 600 650 7000

2×107

4×107

6×107

8×107

1×108

Time (min) post reperfusion

C3b

dep

osit

ion

in

kid

ney

Den

isto

metr

ic s

um

ControlTreated

50 100 150 200 250 300 350 400 450 500 550 600 650 7000

2×107

4×107

6×107

8×107

Time (min) post reperfusion

C4d

dep

osit

ion

in

kid

ney

De

nis

tom

etr

ic s

um

Control

Treated

50 1001502002503003504004505005506006507000

1×108

2×108

3×108

4×108

Time (min) post reperfusion

C5aR

dep

osit

ion

in

kid

ney

Den

isto

metr

ic s

um Control

Treated

50 100 150 200 250 300 350 400 450 500 550 600 650 7000

1×108

2×108

3×108

Time (min) post reperfusionM

AC

dep

osit

ion

in

kid

ney

Den

isto

metr

ic s

um

Control

Treated

*

Complement deposition

** ** **** *** *** *** *** ***

**

*

Coagulation and Complement activation

*** **

*

*

Diuresis

To

tal d

iure

sis

(m

l) d

uri

ng

360 m

in p

ost

tx

Control PEG-phospholipid0

20

40

60

80

Control

PEG-phospholipid

n=6

*

3A

Control PEG-phospholipid

construct

Cytokines

Inflammatory markers

0.1

0.25

0.50 1 24 48 72 96 12

0

0

100

200

300

400

500

Time (hour) post reperfusion

MC

P-1

(p

g/m

l)

Control (n=3)

PEG-lipid (n=7)

0.1

0.25

0.50 1 24 48 72 96 12

0

0

200

400

600

800

Time (hour) post reperfusion

Control (n=3)PEG-lipid (n=7)

TN

F@

(p

g/m

l)

Plasma Creatinine

Kidney function marker

Time (hour) post reperfusion

Pla

sm

a C

reati

nin

e u

mo

l/L

24 48 72 96 120

0

500

1000

1500Control (n=4)PEG lipid (n=7)

*

**

*

Summary

1. PEG/phospholipid-linked regulators are efficientregulators of these reactions in vitro

2. Also the PEG/phospholipid linker alone has a significant effect on I/R injury in vivo

Sana Asif

Karin Fromell

Jaan Hong

Rolf Larsson

Peetra Magnusson

Kristina Nilsson Ekdahl

Uppsala Universitet

Yuji Teramura

Tokyo University/Uppsala University

Marianne Waern Jensen

Swedish Agricultural University, Uppsala

Alireza Bigliarnia

Lund University

Peter GarredCopenhagen University

John LambrisDaniel RicklinPennsylvania University

Markus Huber-LangUlm University, Ulm

Per NilssonOslo UniversityLinneus University

John LambrisDaniel RicklinPennsylvania University

Trian ChavakisIannis KourtzelisDresden TechnicalUniversity

Tack!