Described how a blood coagulation defect leads to impaired wound healing

1. The Effect of Hemophilia on
Wound Healing
Maureane Hoffman, MD, PhD
Professor of Pathology
Duke University Medical Center
Durham, North Carolina

2. Everybody knows that hemophiliacs
have abnormal wound healing.
HR Roberts
(personal communication)

3. Wound healing

4. How does hemostasis
influence healing?
• Platelets release a wide variety of cytokines
and growth factors
• Some activated factors have other biological
activities, including promoting inflammatory
and immune responses
• Fibrin clot provides a framework for tissue
repair and fibrin degradation products are
chemotactic for leukocytes

5. How does hemostasis
influence healing?
• Platelets release a wide variety of cytokines
and growth factors
• Some activated factors have other biological
activities, including promoting inflammatory
and immune responses
• Fibrin clot provides a framework for tissue
repair and fibrin degradation products are
chemotactic for leukocytes

6. How does hemostasis
influence healing?
• Platelets release a wide variety of cytokines
and growth factors
• Some activated factors have other biological
activities, including promoting inflammatory
and immune responses
• Fibrin clot provides a framework for tissue
repair and fibrin degradation products are
chemotactic for leukocytes

7. Thrombin has cytokine and
growth factor-like activities
• Monocyte/macrophage chemotaxis
• Epithelial cell proliferation
• Angiogenesis
• Fibroblast proliferation
Dawes et al. Thrombin stimulates fibroblast chemotaxis and replication. Eur J Cell Biol 1993;
61:126-130.
Algermissen et al. Distribution and potential biologic function of the thrombin receptor PAR-1
on human keratinocytes. Arch Dermatol Res 2000;292:488-495.
Norfleet et al. Thrombin peptide, TP508, stimulates angiogenic responses in animal models
of dermal wound healing, in chick chorioallantoic membranes, and in cultured human aortic
and microvascular endothelial cells. Gen Pharmacol 2000;35:249-254.

8. Hypothesis
Reduced thrombin generation
leads to impairment of the time
course and/or tissue features of
wound healing

9. Experimental model
Control: Wild-type mice C57BL/6 (WT)
Experimental: Coagulation Factor IX-knock-out,
hemophilia B (HB), mice developed in Darrell Stafford’s
Laboratory
Lin HF, Maeda N, Smithies O, Straight DL, Stafford DW. A coagulation
factor IX-deficient mouse model for human hemophilia B. Blood
1997;90:3962-3966.

10. Punch Biopsy Wounds
Hoffman et al Blood 2006; 108: 3053-3060

11. Time course of healing
Hoffman et al Blood 2006; 108: 3053-3060

12. Day 2 - HB mouse
• Untreated HB mice often
develop subcutaneous
hemorrhage
Hoffman et al Blood 2006; 108: 3053-3060

13. Beneath the Surface of a Skin Wound

14. Immunohistochemistry
Fibrin -
MP Influx -
Angiogenesis -
Tissue Iron -
Reduced in Hemophilia

15. The top panel shows an electron
micrograph of a wound from a
hemophilia A subject 2 hours after
wounding, which is filled with red
cells (R) and scattered fibrin (F).
The bottom panel shows a similar
wound from a normal subject with
no red cells, tightly interdigitated
platelet ghosts surrounded by fibrin
fibers.

16. Immunohistochemistry
Fibrin -
MP Influx -
Angiogenesis -
Tissue Iron -
Reduced in Hemophilia
Reduced and delayed

17. Macrophage infiltration into skin
wounds in WT and HB mice
Hoffman et al Blood 2006; 108: 3053-3060
0
0.9
1.8
2.7
3.6
0 5 10 15 20
days
MPscore
WT
HB

18. Immunohistochemistry
Fibrin -
MP Influx -
Angiogenesis -
Tissue Iron -
Reduced in Hemophilia
Reduced and delayed
Increased

19. Hoffman et al Blood 2006; 108: 3053-3060
Angiogenesis is increased in
skin wounds in WT and HB mice
0
5
10
15
20
25
0 2 4 6 8 10 12 14 16 18
Days After Wounding
Vessels/hpf
WT
KO

20. Immunohistochemistry
Fibrin -
MP Influx -
Angiogenesis -
Tissue Iron -
Reduced in Hemophilia
Reduced and delayed
Increased and persists
Increased

21. Iron persists in the tissues even after
healing in HB mice
• Top panel is from a HB
mouse 10 days after
wounding. Extensive iron
staining (blue) is visible in
the deep tissues
• Bottom panel is from a WT
mouse at day 10. Very little
iron staining is seen

22. Iron has been linked to
inflammation and impaired healing
• Yeoh-Ellerton S, Stacey MC. Iron and 8-isoprostane
levels in acute and chronic wounds. J Invest Dermatol.
2003;121:918-925.
• Morris CJ, Blake DR, Hewitt SD, Lunec J. Macrophage
ferritin and iron deposition in the rat air pouch model of
inflammatory synovitis. Ann Rheum Dis. 1987;46:334-
338.

23. Iron has been linked to
hemophilic arthropathy
• Morris et al. Relationship between iron deposits and tissue damage
in the synovium: an ultrastructural study. Ann Rheum Dis.
1986;45:21-26.
• Roosendaal et al. Iron deposits and catabolic properties of synovial
tissue from patients with haemophilia. J Bone Joint Surg Br.
1998;80:540-545.
• Wen et al. c-myc proto-oncogene expression in hemophilic
synovitis: in vitro studies of the effects of iron and ceramide. Blood.
2002;100: 912-916.
• Hakobyan et al. Pathobiology of hemophilic synovitis,I:
overexpression of mdm2 oncogene. Blood. 2004;104:2060-2064.

24. Mouse Model of Joint Bleeding
• Panels show increasing
degrees of new vessel
formation (angiogenesis)
in a hemophilia mouse
model of joint
hemorrhage
– Valentino & Hakobyan.
Haemophilia; 2006. 12:654-
62

25. Take-home message #1:
Hemostasis not only stops bleeding,
it sets the stage for tissue healing.
Inadequate hemostasis can
promote damaging inflammation and
angiogenesis by deposition of iron.

26. Hypothesis:
Restoring the initial level of
thrombin generation will
normalize subsequent wound
healing in hemophilia B

27. Experimental Design
• A single dose of hemostatic agent was given
about 30 min before punch biopsy wound
placement
• Replacement - human FIX to raise level to 100%
• “Bypassing” - human FVIIa to a level that
normalizes the tail bleeding time

28. Wound healing in pretreated mice
McDonald et al J Thromb Hemost 2007; 5:1577-1583

29. Early and Late Hematomas
• WT mice never get hematomas
• HB mice initially get hematomas at the bx site
• Pre-treatment reduces the early hematomas
• Late hematomas still occur away from the wound site!?

30. Take-home message #2:
Restoring thrombin
generation at the time of initial
hemostasis is not enough to
normalize subsequent healing

31. Why do HB mice bleed even after
the surface wound has healed?

32. 1) Bleeding in granulation tissue
• This picture shows
neovessels and hemorrhage
within the granulation tissue
in a well-healed 15d wound
in a HB mouse
• Even WT mice have small
amounts of bleeding in
granulation tissue

33. Traveling the Macrophage Highway
• These views are from a 15d HB
wound. The “macrophage highway”
is just beneath the skeletal muscle.
On the right side is an area
expanded by inflammation,
granulation tissue and fibrosis
• There are many new vessels along
with hemosiderin-laden
macrophages

34. 2) Hematomas occur in the MP Highway

35. Could inflammation or angiogenesis
cause the bleeding in HB mice?

36. “Blister” model of pure inflammation
• Filter paper soaked in 0.05% cantharidin placed on skin
• Mouse sacrificed and skin collected 24 or 48 hr later
• This protocol leads to a blister in humans (we don’t
usually do the “sacrifice” part in humans)
• Mice respond differently - we see acantholysis, but no
fluid collection to form an actual “blister”
• After a few days the epithelium regenerates with no
scarring and no angiogenesis

37. Cantharidin Application - day 2
We see intra-
epithelial separation
in top panels,
with extensive
infiltration of PMN
into dermis and
epidermis (lower
left).
To get there PMN
marginate and
migrate through the
vessel wall (lower
right)

38. Cantharidin Application - Hemophilia B Mice
Tissues from 2 day
old wounds in HB
mice show the same
histologic features as
WT, including WBC
influx.
The only difference
is occasional RBC
extravasation (as in
the lower right).

39. Macrophage influx after cantharidin
• Significant macrophage influx is seen
(brown color) in this section from a HB
mouse 2 day after cantharidin
• WT mice looked similar
• A high power view of the “blister” area
(intraepidermal separation) shows that
many of the cells in this area are
neutrophils

40. What about in thrombocytopenia?
Thrombocytopenic mice have been
reported to bleed during inflammation
induced by croton oil application.
Goerge et al: Inflammation induces hemorrhage in
thrombocytopenia. Blood, 111:4958-64, 2008

41. Even without inflammation,
thrombocytopenia leads to
endothelial changes
• This picture shows a red cell
extravasating from a capillary
of a thrombocytopenic rabbit
• RBC appear to traverse small
channels in the endothelial
cells.
• Aursnes & Pedersen:
Petechial hemorrhage in
the ciliary process of
thrombocytopenic rabbits.
An EM study.
Microvascular Res, 17:12-
21, 1979

42. Thrombocytopenic mice also bleed in the
cantharidin blister model of inflammation
Hemorrhage is present
in and around the
“macrophage highway”
in skin sections from wild
type mice rendered
thrombocytopenic with
anti-GPIb anti-serum at
the time of cantharidin
placement

43. Take-home message #3:
Hemophilia B mice have a
normal leukocyte response in the
blister model
Thus, different mediators are
involved in leukocyte responses in
the wound and blister models

44. Take-home message #4:
Inflammation alone causes
bleeding in thrombocytopenia,
but does not cause bleeding
in hemophilia
This tends to support the hypothesis
that angiogenesis predisposes to
bleeding

45. • Disruption of the structure of existing
vessels as angiogenic sprouts develop
• The delicate and leaky nature of the new
vessels
• Lack of TF procoagulant expression in and
around angiogenic vessels
• Mc Donald et al. Perivascular tissue factor is down-regulated following
cutaneous wounding: implications for bleeding in hemophilia; 2008
Blood 111:2046-9
Angiogenesis during healing
predisposes to bleeding due to:

46. Hypothesis
Could late hemorrhage in hemophilia B mice
(and recurrent joint haemorrhage in humans)
be due to a vicious cycle…
…whereby…
…iron deposited by bleeding stimulates
increased angiogenesis, which then
predisposes to more bleeding?

47. Speculation
• Stopping bleeding is never as good as
preventing it in the first place
• Injury could trigger the cycle of
bleeding/angiogenesis/more bleeding

48. Thanks to:
• Mac Monroe, PhDMac Monroe, PhD
• Harold Roberts, MDHarold Roberts, MD
• Alisa Wolberg, PhD
• Julie Oliver, PhD
• Anna McDonald, MD
• Zhi Hong Meng, MPH
• Angie Lenkowski
• Jacqueline Brock
• Ulla Hedner, MD
• Support from the American Heart Assn,
NHLBI, Novo Nordisk A/S and the US
Department of Veterans Affairs

49. Multiday treatment - VIIa