1. Hemoglobin Disorders and the Sickle
Hemoglobinopathies as
Inflammatory and Thrombotic Disorders
Babette B. Weksler MD
October 16, 2014
No conflict of interest to disclose

2. Hemoglobin Abnormalities
1. Quantitative defects: imbalance of chain
synthesis  thalassemia syndromes
2. Qualitative defects: additions, substitutions or
deletions of amino acids e.g. sickle cell disease
or altered oxygen affinity, stability
3. Failure to silence genes: hereditary persistence
of fetal hemoglobin (HPFH)

3. Hemoglobin Genes and Products
Hgb is a tetramer of the products of two genes,
alpha globin and non-alpha globin.
2 identical a chain genes on chromosome 16p
g, d, b chain genes clustered on chromosome 11p
Hemoglobin Genotype Amount
Hb A a2b2 96%
Hb A2a2d2 3%
Hb F a2g2 1%
If a-chain synthesis is decreased, see Hb Barts = g4 or Hb H =b4

4. Two Human Globin Gene Clusters
Exist on Different Chromosomes
FETAL
α2γ2
F
ADULT
α2δ2 α2β2
A2 A
EMBRYONIC
α2ε2 ζ2ε2 ζ2γ2
Gower 1 Gower 2
Portland

5. Timeline and Sites of Synthesis of the
Different Hemoglobin Chains

6. Case Presentation
History:
20 yo Afro-American male
10 year H/O leg ulcers, otitis media,
pneumonia, and attacks of abdominal pain
with jaundice
1 prior episode joint swelling and pain
Presented with fever, cough and anemia
Physical Exam:
pallor, scleral icterus, round and oval scars over
lower extremities
bilateral rales, dullness both bases
cardiomegaly with soft systolic murmur

7. Case Presentation, continued
Urinalysis: trace albumin; few granular casts
Peripheral blood: WBCs 15,250/mm3
72 P, 1 Band, 15L, 7 M, 5 Eo
RBCs 2,800,000/mm3
74 nRBCs/100 WBC

8. Case Presentation, continued.
This was the first description of
sickle cell anemia, 100 yrs ago in
a dental student from Granada
who presented with pneumonia to
Cook County Hospital in Chicago
in 1909 on Christmas eve.
James R. Herrick “Peculiar
elongation and sickle-shaped red
blood corpuscles in a case of
severe anemia.” Arch Int Med
6:517, 1910.
“…some change in the composition
of the corpuscle itself may be
the determining factor”

9. Itano, Singer, Wells, and Pauling - 1949
demonstated abnormal electrophoretic
mobility of Hb S and predicted molecular
charge alteration

10. A single invariant point mutation in the beta globin
gene is the basis for sickle cell disease
GAG GTG
bglu6 val6
Yet the phenotype is extremely variable

11. Linus Pauling’s 1949 Prediction of
Molecular Basis of Sickling
"Let us propose that there is a surface region on the . . . sickle cell
anemia hemoglobin molecule which is absent in the normal molecule
and which has a configuration complementary to a different region of
the surface of the hemoglobin molecule. . . Under the appropriate
conditions [as in low oxygen or air pressure], then, the sickle cell
anemia hemoglobin molecules might be capable of interacting with one
another at these sites sufficiently to cause at least a partial
alignment of the molecules within the cell, resulting in the
erythrocyte's . . . membrane's being distorted to accomodate the
now relatively rigid structures within its confines.”
Longitudinal “liquid
crystal”
Cross section of
crystal stack

12. Hemoglobin Structure and Hb S
Mutation b6 GluVal
induces charge changes
on surface of Hb
molecule, favoring
aggregation when O2

13. Hemoglobin S molecules stack together
when deoxygenated
Heme groups = red, mutant valines = blue

14. Peripheral Blood

15. Rheologic effect of intravascular sickling

16. Natural History of Sickle Cell Disease
1960’s Disease of childhood
1973 Median age at death = 14 yr (1/6 cases>50)
1990 Survival >20 yr of 85%, if SC 95%
1994* Median age at death 42 for men, 48 for women
2001 Median age at death 53 for men, 58 for women
Main causes of death: organ failure 18%, stroke
22%, acute event 32%
* when hydroxyurea became available

17. Mortality Rates for Adults and Children
(<19yr) with SCD in the US 1979-2005
1980 1985 1990 1995 2000 2005
3
2.5
2.0
1.5
1.0
0.5
Mortality rate per 100,000 Afr-Amer
population
Adult
Child
Lanzkron, Pub Health Repts 128:110, 2013

18. Predictors of Increased Mortality in
Sickle Cell Disease
Persistent high WBC and platelet count
Acute chest syndrome
Pulmonary hypertension
Renal impairment
Hb < 7 g/dL

19. Bone infarcts and marrow expansion in SCD
Fishbone vertebrae Infarction of humeral head

20. Functional Asplenia
5 months 7 months

21. Stroke in SCD

22. Acute Chest Syndrome in SCD

23. Sickle cell ulcer

24. Modulating Factors in Sickle Cell Disease
High Hb F (interferes with sickling)
Other non-S hemoglobins
Beta thalassemia trait/ alpha thalassemia trait
Beta globin haplotype: Senegal>Benin>Bantu>Cameroon
(correlates with Hb F levels and to X-linked F-cell
production locus)
Genes that modify Hb class switching:
e.g. BCL11A, silences F Hb production
r57482144, promotes F Hb production

25. Polymerization Delay Times in Human Hemoglobin Mixtures
HbS= 100% S; HbF= 75%S. 25%F; HbAS3= 75%S. 25%AS3
Levasseur JBC 2004

26. a-Thalassemia: 1 or 2 Gene Deletion
if combined with SS, ameliorates phenotype
by decreasing MCH

27. Sickle/Beta+ Thal has milder phenotype due to low
MCV, residual Hb A and lower Hb S content but
Sickle/Beta-zero Thal is as severe as SS

28. Hemoglobin SC Disease
% Hb S is greater than in sickle trait, so more severe
Crises less frequent than SS, Hb level higher
and ranges from ~8 g/dL to normal.
RBC lifespan longer than in SS
RBC dehydration
High rate of aseptic necrosis and retinal disease
Splenomegaly common
Anemia, crises common in pregnancy
Responds to hydroxyurea

29. Target Cells in Hb CC
56 yo AA man, Hb 10.8, MCV 78, normal LFTs, spleen
palpable

30. Inflammation and Enhanced Coagulation Are Keys to
Sickle Cell Pathology
Vaso-occlusion Hemolysis
RBC Sickling
Tissue
Factor
EC
dysfunction
Inflammation Thrombin Generation
ROS Integrins PMN-RBC aggregates Microparticles

31. Inflammation in SCD
Hypoxia  RBC sickling  Vaso-occlusion 
Endothelial dysfunction  prothrombotic EC
 integrin expression
 NO deficiency
 Increased adhesion
of RBC, platelets, WBC
 EC microparticles
…..further occlusion, sickling, hypoxia,
and organ damage

32. Coagulopathy in SCD
Activated vascular endothelium = prothrombotic
Decreased vascular NO favors platelet activation
Prothrombotic microparticles from RBC and platelets
Increased blood tissue factor level
VWF activating factor increased (Activated A1 domain
that binds platelets)
Total VWF increased
High WBC and positive surface PS also favor thrombosis
Pulmonary hypertension favors silent pulmonary emboli

33. Inflammation and Enhanced Coagulation Are Keys to
Sickle Cell Pathology
Vaso-occlusion Hemolysis
RBC Sickling
TF
Inflammation Thrombin Generation
ROS Integrins PMN-RBC aggregates Microparticles
vascular NO depletion S-1P

34. Increased Coagulation during VOC in Sickle Cell Disease
Inpatient=VOC Outpatient= steadu state
D dimer 3077+ 3655 D dimer 1041 + 619
Shah Thr. Res. 130:3241,2012

35. RBC Microparticles Increase in Stored Blood and Can
Scavenge Vascular Nitric Oxide, Favoring Vasoconstriction
and Platelet Activation
Red cell microparticles react with the important signaling
molecule NO almost as fast as cell-free hemoglobin, about
1000 times faster than red-cell-encapsulated hemoglobin
Liu, Free Rad Biol Med 65:1164, 2013

36. Although Sickle cell disease was the first molecular
disease described, very few controlled clinical
studies have been done re pathophysiology.
Trials showed the only proven effective treatments
are penicillin and hydroxyurea, plus transfusion.
BMT is the only curative therapy.
Transgenic mice bearing all human hemoglobin HbA
or Hb S (Berkeley and Townes mice) now permit
direct studies of vaso-occlusion and hemolysis and
testing of new therapeutics

37. Prothrombotic State in SCD Leads to Inflammation
via TF-dependent FX activation
Sickling produces TF activation via 2 mechanisms: hemolysis or
WBC/inflammatory mechanisms
Activated monocytes/PMN or microparticles express TF 
prothrombotic effect and increase soluble VCAM
Endothelial TF procoagulant activity is limited by TFPI and EPCR
whille EC TF signals via protease-activated receptor-2 (PAR-2) to
release IL-6
Thus Fxa selective anticoagulants may limit both microthrombosis
and proinflammatory IL-6 in SCD, with less hemorrhagic potential
Studies carried out in SCD mice
Sparkenbaugh, Blood 123:1747, 2014

38. Tissue Factor (TF) Plays Central Role in
Vascular Inflammation/Thrombosis in SCD
Ruf W Blood 2014;123:1630-1631
©2014 by American Society of Hematology

39. Tissue Factor (TF) Plays Central Role in
Vascular Inflammation/Thrombosis in SCD
Ruf W Blood 2014;123:1630-1631
©2014 by American Society of Hematology
Free heme
Abnormal rbc membrane
Activated platelets
all augment thrombin
generation

40. Tissue Factor (TF) Plays Central Role in
Vascular Inflammation/Thrombosis in SCD
Ruf W Blood 2014;123:1630-1631
©2014 by American Society of Hematology
Free heme
Abnormal rbc membrane
Activated platelets
all augment thrombin
generation
Endothelial TF and activated EC
Release circulating IL-6

41. Proposed role of FXa and thrombin in vascular inflammation in SCD. FSP, fibrin split product.
Sparkenbaugh E M et al. Blood 2014;123:1747-1756
©2014 by American Society of Hematology

42. New NIH Consensus Recommendations for
Management of Sickle Cell Disease
Children: Daily penicillin to age 5 yr early pneumovax
Yearly transcranial Doppler age 2-16 for SCD
Chronic transfusions for TCDV >200 cm/s
Children and Adults:
Opioids, fluids & Spirometry for vaso-occlusive crisis.
Physical therapy, analgesia for avascular necrosis
ACE inhibitors for microalbuminemia
Laser Rx for proliferative SCD retinopathy
Echocardiography to evaluate for pulmonary hypertension
Hydroxyurea for SCD adults with >3 VOC/yr or ACS
Pre-op transfusion to bring Hb to 10 g/dL, Hb S<30%
Assess iron overload and use Fe chelation
Yawn P et al JAMA 312(10) 1033, 2014

43. Mainstays of Therapy in SCD
Analgesics
Fluid, Oxygen
Blood
Immunizations
Penicillin
Hydroxyurea
New approaches: anti-inflammatory and
anti-thrombotic therapies

44. Hydroxyurea in Sickle Cell Disease
Raises Hb F, decreases Hb S polymers in rbc
Decreases sickling, crises, ACS
Decreases inflammatory cytokines
Lowers WBC
Induces eNOS-cGMP pathway in endothelium
Reduces procoagulant state of endothelium
Decreases abnormal angiogenesis
Decreases stroke incidence in SCD children

45. Longterm Adult Use of Hydroxyurea in SCD
median use >10 yr Hb F rise 2-11x baseline
No HU HU
Crises/yr 7.4 + 6.5 0.2 + 0.4
Tx/yr 1.5 + 5.9 ~0
Hosp/yr 2.1 + 2.9 0.6 + 0.2
ACS 6.1% 0.8%
OS 65% 86%
Stroke 5% 2.5%
AVN No difference
Pulmonary HT No difference
Voskaridou, Blood 115:2354, 2010

46. Transfusion in SCD
High level matching to avoid alloimmunization
Rh, Kell, Duffy very important
Exchange vs simple transfusion
Blood viscosity (more is not better)
Chronic transfusion in children at risk of stroke
Transfusion clearly better than switch to hydroxyurea
Pre-operative transfusion
Goal to reduce Hb S to 30%, raise Hb to 10, no sig difference
Transfusion in pregnancy
No controlled trials
Iron overload management—Exjade
in SS, less tissue iron deposition than thal; deferiprone also
useful

47. Is Gene Therapy Feasible for SCD?
? Use of autologous CD34 or IPS cells
Need for safe and effective vector
Requirement for LCR as well as coding
region for human BA-globin gene
Harvest sufficient HSC from recipient
Stable expansion of transduced HSC

48. HbAS3 Gene Therapy Corrects Sickling in Sickle Mice
Levasseur JBC 2004 Recipient mice expressed 20-30% HbAS3

49. Cross Section of Polymerized Hemoglobin SS Fiber

50. Antisickling Properties of Mutated Hb bglobin Chain
Levasseur JBC 279:27518, 2004 Fig 1
bglu87
decreases
polymerization
bala22 decreases
polymerization
basp16
stabilizes
a/b interaction

51. Lentiviral Provirus Carrying Modified Beta Globin Casette
Human Hb b globin gene modified at 3 sites to impede sickling
b globin locus control region
FB insulators

52. Erythroid Differentiation of Transduced CD34+ Cells
from Healthy Donors (HD) or Sickle Cell Donors (SCD)
Romero JCI 123:3319, 2013

53. Deoxygenated RBC from HBBAS3-Transduced SCD CD34+ Cultures
Romero JCI 123:3319 Fig 5

54. In Vivo Detection of Engrafted HBBAS3-Transduced
CD34+ Cells Transplanted into NSG Mice
SCD SCD HD HD SCD HD
Romero JCI:123,3319;’13 Mock HBBAS3

55. Adenosine A2A Receptor Agonists for
Inflammation Control in SCD
During VOC, NFKb p65, IFN, A2AR are elevated in iNKT
cells (0.5% of all T cells)
Regadenoson (Lexiscan) an A2AR agonist, is FDA
approved for myocardial perfusion studies
Low dose Regadenoson infusion x 24 h in SCD patients
lowers iNKT inflammatory markers to normal during VOC
Mechanism is via deactivation of iNK-T cells
No toxicity
21 pts steady state, 6 VOC reported in Blood 4/25/13
Phase II study started April 2013. Clin Trials Gov
#NCT1788631

56. Selectin Inhibitors to Treat
Vasoocclusive Crisis in SCD
GMI 1070, E-selectin inhibitor, restores blood flow in mouse
model of SCD.
Phase I human study in steady state SCD patients shows GMI
1070 is safe, decreases markers of endothelial activation,
WBC activation, and clotting

57. What’s New and Hopeful in SCD Management
Better understanding of inflammatory and
procoagulant pathophysiology” Factor Xa inhibitors
Promising, Voraxapar NOT
Better management of pediatric SCD-transcranial
doppler, transfusion programs, early hydroxyurea
New concepts about role of hydroxyurea
Microfluidic techniques for probing rheology
Better grasp on how to raise HbF
Novel pharmacologic approaches

58. Is there anything positive about Hb SS red cells?

59. Is there anything positive about Hb SS red cells?
HOW ABOUT CANCER CHEMOTHERAPY?
Take advantage of the propensity for Hb SS
rbc to sickle in hypoxic environments, obstruct
microvessels and damage endothelium……

60. Figure 8. Schematic depiction of proposed pathophysiology of tumor killing induced by SSRBCs and the HO-
1 inhibitor ZnPP.
Terman DS, Viglianti BL, Zennadi R, Fels D, et al. (2013) Sickle Erythrocytes Target Cytotoxics to Hypoxic Tumor
Microvessels and Potentiate a Tumoricidal Response. PLoS ONE 8(1): e52543. doi:10.1371/journal.pone.0052543
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052543

61. SS RBC Home to Hypoxic Tumor Vessels and Destroy
Tumor in Mouse Mammary Cancer Model
Terman D, PLoS One i8:e52543, 2013

62. Sickle RBC Home to Hypoxic Tumor Vessels,
Deliver Cytotoxic Molecules and Prolong Survival

63. Sickle Hemoglobinopathies in 2014
Slow increment in lifespan with supportive care and hydrea
Marked improvement in childhood morbidity
Almost no specific drugs: folate, penicillin, hydrea, rbc
Improved understanding of pathogenesis of crisis involving
inflammation and hypercoagulability
Better prenatal diagnosis possible
Slow progress in gene therapy
Minimal progress in management of SCD in poor countries