Pathophysiology: Hematology

Hematopoesis ......................................................................................................................................................................... 2
Consequences of & Approach to Anemia ............................................................................................................................... 4
Iron Deficiency Anemia ........................................................................................................................................................... 6
Folate & B12: Metabolism & Deficiency .................................................................................................................................. 9
Hemolytic Anemia ................................................................................................................................................................. 12
Hemoglobinopathies ............................................................................................................................................................. 16
Hemostasis ............................................................................................................................................................................ 20
Thrombosis............................................................................................................................................................................ 25

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 Hematopoesis
Hematopoeisis (Gr. haimato, “blood” + poiesis, “creation”): The formation of blood cells in the living body

Stem cells: can self-renew (proliferate) or differentiate

 Totipotent: can regenerate entire organism (incl. extraembryonic tissues)
 Pluripotent (e.g. embryonic stem cells): can regenerate across germ layers (no extraembryonic tissues)
 Multipotent (e.g. adult stem cells): can regenerate cell types restricted by germ layer
Zen thought of the lecture: Every time a stem cell divides, it’s still a stem cell but a little less so (not stochastic)

HEMATOPOETIC STEM CELLS (2 classes) OTHER PLAYERS (IN STROMA)

Most primitive Myeloid Growth factor/cytokine Stimulates…
(“ high quality”) (“low quality”) Erythropoietin (kidneys) RBC
Precursor to: All lympho-hematopoetic Granulocytes, RBCs, Thrombopoietin (liver) Platelets, HSC
lineages platelets, B-cells(?) Flt-3 Dendritic cells, HSC
Disease Rarely involved Most “stem cell disorders” Stem cell factor Mast cells, HSC
Engraftment Delayed but life-long Rapid but limited G-CSF PMNs
CD34+/-, other markers CD34+, other markers +,
Phenotype
mostly -, smaller larger
Aldehyde DH +++ + (or low)
Note on engraftment: both progenitors & primitive (high quality) stem cells can give rise to all elements; the difference is
in how long they can reproduce (lose graft after initial good result with low-quality stem cells)

Glossary
 Clone: cell population derived from single ancestral cell

 CFU: Colony-forming unit: represents the cell that gives rise to a colony (assayable growth in vivo / in vitro)
o E.g. CFU-S, spleen CFU (mouse low-quality hematopoietic stem cells, give rise to spleen colonies post-BMT-irradiation)
 Colony-forming assay: isolate mononuclear cells from marrow; let ‘em grow (clones)

 CFU-GM: colony—forming unit granulocyte macrophage: most differentiated myeloid progenitor, no self-renewal
o White colonies on assay: makes white blood cells
 BFU-E: burst-forming unit erythroid: RBC progenitor
o Red colonies on assay; making RBCs
 CFU-Mixed / CFU-GEMM: progenitor of both CFU-GM and BFU-E (probably like CFU-S)
o Mixed coloration on assay; making WBC & RBC too

Malignancy
Malignancy: unregulated clonal growth; from 1+ mutations
Pathways to malignancy
Cancer stem cells: (KNOW THESE)
 tumor initiating cell with limitless self-renewal, limited differentiation  increased proliferation
 Treatments often don’t target them  block in apoptosis
(more common)

Leukemic stem cells: mostly at low-quality HSC (myeloid precursor)

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 Acquired Aplastic Anemia
 Hypocellular bone marrow, severe pancytopenia, often in young patients
 Autoimmune disorder: CTLs target low-quality stem cells
o Reduced CD34 stem cell pool  pancytopenia
Pathways to bone marrow failure
 Treatment: cyclophosphamide,
(KNOW THIS)
o activated in liver, blows away lymphocytes
1. Oncogenesis / mutations
o stops T-cell reaction against low-quality stem cells (leukemia, MDS, dyskeratosis congenita)
o High-quality stem cells are saved (have aldehyde DH, 2. Direct DNA damage
inactivate cyclophosphamide) (radiation, benzene, chemo)
3. Autoimmunity
Chronic myeloid leukemia (aplastic anemia, pure red cell aplasia)
 BCR-ABL fusion protein (t9:22 – Philadelphia chromosome) blocks 4. Viruses
(HIV, parvovirus)
apoptosis
 Called a “myeloproliferative” disorder – is really a “ myelo-accumulative disorder”

Diseases of Hematopoesis & where they come from

Notes about this crazy picture (down = more differentiation)

Top of the pyramid: high quality / primitive hematopoetic SC.

 Few diseases at this level
Next: low quality / myeloid SC.
 MOST DISEASES here.
Third: lineage-committed progenitors (a bit more differentiated; like those colony-forming units).
 Pediatric diseases are in this category; possibly why they do better clinically (more differentiated)
Bottom: mature / differentiated blood cells.
 Autoimmune diseases attack these more differentiated cells; lymphomas come from here

Final random thoughts:

 You can make human universal stem cells by transducing 4 genes into adult skin cells.
 Remember: epigenetics is important too

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 Consequences of & Approach to Anemia
Metabolic / Physiologic Responses to Anemia

O2 Delivery & Uptake: 𝑉𝑂2 = 1.39 × 𝑄 × 𝐻𝑏 × (𝑆𝑎 𝑂2 − 𝑆𝑣 𝑂2 ) VO2 = oxygen delivery

1.39 = constant (mL O2 that bind to 1 gm Hb)
Ways to increase oxygen delivery Q = blood flow (mL/min)
1. Increase Blood Flow (Q) Hb = hemoglobin (g/dL)
a. ↑ Cardiac output: ↑HR, ↑pulse pressure, murmurs, bruits, SaO2 -SvO2 = A-V % sat difference
hyperdynamic precordium, tinnitus/roaring in ears (ability to unload oxygen)
b. Change tissue perfusion
i. shift from O2-insensitive (skin: pallor, kidney)  O2 sensitive (heart, brain, muscles)

2. Increase Red Cell Mass (Hb)

a. ↑ EPO (kidney)  Reticulocytosis (see ↑ immature RBCs)
i. Usually lose 1% RBC/day so reticulocyte count is 1%;
higher = “reticulocytosis”
ii. Clinical finding: bony pain, expansion of marrow (e.g. on
imaging)
b. Increased hematocrit is good: to a point!
i. Increase too much: viscosity increases; overwhelms
increased ability to transport; oxygen transport actually
decreases!
ii. Hypervolemia helps a bit (increase volume, can fit more
RBC in) but still can be overwhelmed

3. Increase oxygen unloading (SaO2 -SvO2)

a. ↑ 2,3- DPG (decreased affinity: displaces oxygen from hemoglobin)
i. Generated from glycolytic pathways (anaerobic)
ii. RBC have mostly anaerobic metabolism (90%, 10% aerobic)
1. Allows RBC to generate ATP (maintain shape, flexibility, cation/H2O balance)
iii. More in women (better oxygen delivery)
b. General characteristics of oxyhemoglobin dissociation curve
(see below)

Oxyhemoglobin Dissociation Curve

Oxygen affinity (P50): P50 varies inversely with oxygen affinity
 partial pressure of oxygen when Hb 50% saturated
 curve shifted to right: ↑P50, ↓oxygen affinity
 curve shifted to left: ↓P50, ↑oxygen affinity

INCREASED… SHIFTS CURVE TO… OXYGEN AFFINITY… BECAUSE

You’re bumping O2 off to deliver more to tissues (which is
2,3 DPG Right ↓Decreases
why your kidneys are cranking out 2,3 DPG in the first place)
Bohr effect: if carbon dioxide rises in a tissue, you need more
pH Left ↑Increases oxygen. Blood gets more acidic from CO2, pH drops, oxygen
affinity decreases, and more O2 gets dropped off
Oxygen Left ↑Increases You’ve got high O2 – why not hang on to it?
If it’s cold, your metabolism slows down, so you don’t need as
Temperature Right ↓Decreases
much O2

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Cooperativity:
 when Hb partially saturated, affinity of remaining hemes increases markedly
 2 Hb conformations: Tense (deoxy) & relaxed (oxy)
 Pictures like the one to the right are popular when discussing cooperativity

Classifying Anemia
1. Cause: is there decreased RBC production, increased RBC destruction, or RBC loss (bleeding)
2. RBC Size: microcytic / normocytic / macrocytic
3. Hb: hypochromic (↓Hb/RBC),normochromic (normal Hb/RBC)
4. Morphology: normal / abnormal (anisocytosis = varied morphology)

Clinical Tests & Definitions

QUESTION TEST
Is the patient anemic? CBC, Hb, Hct
RBC production/destruction/loss? Reticulocyte count (usually ~1%)
Micro/macro/normocytic RBC Indices
Hypo/normochromic
Morphology Peripheral blood smear
PCV g Hb
Hct = ; PCV = packed cell volume (packed RBC volume) Hb =
Vblood dL blood

Hct
Mean Corpuscular Volume: MCV = reflects average size / volume of RBC (in fl, femoliters)
RBC Count

Hb
Mean Cell Hemoglobin: MCH = RBC Count reflects weight of Hb in average red cell

Hb
Mean Cell Hemoglobin Concentration: MCHC = indicates concentration of Hb in average red cell (%)
Hct
Reticulocyte = young RBC
Normal morphology: donut shape, center pallor 1/3 of red cell

Common causes for various types of anemia

Hypochromic, microcytic Normocytic, normal morphology
 Iron deficiency  Hemorrhage / blood loss
 Thalassemia syndromes  Unstable hemoglobins
 Sideroblastic anemia, transferring deficiency  Infection / inflammation / chronic dz
Macrocytic Normocytic, abnormal morphology
 Megaloblastic anemias (folic acid / B12 deficiencies)  Hemoglobinopathies (SS, SC, CC)
 Liver disease, reticulocytosis  Hereditary spherocytosis
 Bone marrow failure syndromes, drugs (AZT, etc)  Autoimmune hemolytic anemia; enzymatic deficiencies

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 Iron Deficiency Anemia
Background: Iron Metabolism
 Distribution: mostly active use (60% Hb, 13% Mb / enzymes)
o also stored (ferritin/hemosederin, 27%); in transport (transferrin 0.1%)
 Intake: 10-25mg from food per day
o Most dietary intake is nonheme iron (spinach, etc) but less bioavailable than heme iron (veal, meat)

From food to blood: lumen mucosal cell blood

(remember that Fe is very oxidative / dangerous & body needs protection from it) Fe Fe
Fe
1. Absorption: brush border of upper small intestine via transport proteins Tf ferritin
sTf
2. Transport: Binds to apotransferrin in mucosal cell  forms transferrin Fe
exported to blood (intracellular apotransferrin recycled)  exported to Apo
blood  bound to soluble transferrin in blood Tf
3. Uptake: cells that need iron have transferrin receptors, ApoTf
di FeTf

e.g. erythroid precursors TfR

4. Storage: mostly in Mϕ of reticuloendithelial system mono FeTf

(liver/spleen/marrow)
o Ferritin: PRINCIPAL IRON STORAGE PROTEIN. Multi-subunit;
form shell around Fe molecules. Serum ferritin is proportional to
intracellular ferritin (lab test). Good for quick mobilization.
o Hemosiderin: insoluble ferritin (packed together until it
precipitates; can see on microscopy. Longer-term storage
Iron cycle:
 Erythroid precursors: uptake via Tf receptors  incorporate
Fe into hemoglobin & package into RBC
 Also used for myoglobin in muscle
 Old RBC destroyed Mϕ pick up iron
o store as ferritin/hemosiderin (recycling)
o release as transferrin into plasma when needed

Pathophysiology of Iron Deficiency

1. Iron stores depleted

2. Fe becomes limiting factor in heme biosynthesis
3. ↓ heme  ↓hemoglobin assembly
4. ↓Hb  ↓ RBC production
5. Small RBC (microcytic / low MCV) & Hb-deficient RBC (hypochromic / low MCH)

Responses to iron deficiency:

1. Increase absorption, transport, uptake of iron (more absorption, more Tf/TfR made)
2. Decrease storage & utilization (less ferritin, microcytosis, hypochromia)

Molecular Mechanisms:
 When plenty of iron is around:
o transferrin mRNA made but unstable (↓Tf protein, ↓transport)
o ferritin provides iron storage; heme synthesis (ALA synthase) uses iron
o Fe bound to IRPs, IRPs inactive (see below)
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 Iron sufficient state:
 Iron deficiency: Iron response proteins (IRP-1 & 2)
ACONI- Fe+++
o IRP-1, IRP-2 lose their iron atoms (↓iron around); start RNA- TASE-1

binding (bind iron response element: IRE)

IRE
o Bind IRE in transferrin mRNA & stabilize  ↑transferrin TRANSFERRIN mRNA (unstable)
transferrin

production (↑iron transport) IRE

FERRITIN mRNA
ferritin
o Bind IREs in ferritin / ALA synthase mRNAs & block translation IRE heme
start site ALA SYNTHASE mRNA

o IRP-1 is aconitase (cytoplasmic TCA cycle enzyme); loses Iron deficient state:
enzymatic activity if no iron (increasing iron for it’s own use!) ACON-
ITASE-1
enzyme inactive;
becomes IRP
IRP
IRE transferrin
Iron sufficient state Iron deficient state TRANSFERRIN mRNA (stable)
IRP
Aconitase TCA enzyme activity IRP-1 functions IRE
ferritin
FERRITIN mRNA
mRNA unstable IRP stabilizes mRNA
Transferritin IRP
↓ transport ↑ transport IRE
ALA SYNTHASE mRNA
heme
mRNA transcribed IRP blocks transcription
Ferritin
↑ storage ↓storage
mRNA transcribed IRP blocks transcription
ALA synthase
↑ heme synthesis ↓heme synthesis

Iron losses
 Iron closely conserved in humans: NO PHYSIOLOGIC MEANS TO EXCRETE EXCESS IRONS
 Very small amounts lost (urine/bile/sweat, cells shedding from GI/urinary tracts; 0.05% Fe lost/day)
 Higher loss states: menses in post-pubertal females, pregnancy (to fetus), lactation (to breast milk)

Pathogenesis of iron deficiency

 Iron deficiency = deficit in total body iron: requirement > supply (intake+storage)

Causes: General symptoms (all anemias)

 recurrent / chronic / occult blood lost (e.g. GI bleed)  Pallor
 failure to meet physiological requirements (rapid growth, menses /  Fatigability, weakness
pregnancy / lactation)  Dizziness
 Inadequate intake (diet low in heme iron, e.g. strict vegans; GI disease,  Irritability
surgery, excessive milk intake in infants)

ALL SIGNS AND SYMPTOMS DEPEND ON: OCCASIONAL FEATURES OF IRON-DEFICIENCY ANEMIA
pagophagia craving ice
 DEGREE OF ANEMIA
pica craving of nonfood substances
 RATE OF DEVELOPMENT OF ANEMIA (dirt, clay, laundry starch)
glossitis smooth tongue
Lab findings angular stomatitis cracking of corners of mouth
Peripheral smear: koilonychia thin, brittle, spoon-shaped fingernails
 Microcytic & hypochromic blue sclerae
 anisocytosis (variable sizes) & poikilocytosis (variable shapes), ovalocytes / eliptocytes

Serum ferritin: LOW (best general indicator of IDA), remember that this is proportional to ferritin in cells
 Serum iron (iron saturation transferrin): expect low iron, high transferrin; ratio is less reliable
Bone marrow iron stain is gold standard (but only used in difficult cases)

CBC / RBC indexes:

 WBC normal with low Hb, low Hct
 Low MCV (microcytosis), low MCH
 Platelet ct: normal/high, retic % low/normal, occasionally high, abs. retic ct low (others are better indicators)
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Sequential changes in lab values
Response to therapy
1. Ferritin decreases: stores being depleted
Peak reticulocyte count 7 - 10 days
2. Iron saturation (iron/transferrin) decreases: you’re iron deficient!
Increased Hb and Hct 14 - 21 days
3. MCV, Hb, Hct decrease: anemia! Hb production is limited now Normal Hb and Hct 2 months
Generally, microcytosis develops before significant anemia Normal iron stores 4 - 5 months

Therapy: RBC transfusion if severe; mostly iron salts (ferrous sulfate) po (IV if required).
 Phytates (cereal grains), tannins (tea), antacids can inhibit Fe absorption; vitamin C (ascorbic acid) helps it

Correcting iron deficiency: need to ID & TREAT the UNDERLYING CAUSE

 GI blood loss (ulcer/tumor parasite); excessive menstral loss (tumor / bleeding disorders),
 Rare conditions (pulmonary hemosiderosis, paroxysmal nocturnal hemoglobinuria)

Differential diagnosis of IDA

 Thalassemia trait: imbalance of globin chain reduction. Also microcytic / hypochromic but iron tests normal
 Anemia of chronic disease: decreasing Fe utilization with adequate stores
o Blood tests can look like IDA but usually ferritin / transferrin normal)

Anemia of Inflammation (Anemia of Chronic Disease)

Causes:
 Chronic Infections: osteomyelitis, pneumonia, abscesses, bacterial endocarditis, meningitis,
HIV, fungus, mycobacteria
 Autoimmune disease - lupus, rheumatoid arthritis
 Malignancy - Hodgkin disease, lymphoma, metastatic carcinoma, sarcoma, multiple myeloma
 Other chronic diseases - congestive heart failure, liver disease, inflammatory bowel disease

Pathophysiology: want to hide iron from bacteria!

1. Inflammation  IL-6 release (endothelial/Kupffer cells)
2. IL-6: makes hepatocytes release hepcidin
3. Hepcidin: inhibits intestinal iron absorption & iron release from Mϕ that
have taken up old RBC Iron deficiency vs. Anemia of Inflammation

IDA Inflammation Both

DDx vs IDA can be difficult: Ferritin ?
 ferritin increases in ACD (want to store more iron) Serum Iron
 total iron binding capacity ( transferrin) increases in IDA (transport more) TIBC ?
 Gets even crazier if both present at once sTfR No 

Marrow Iron No 

Iron Overload Syndromes

 Remember: no route for iron excretion
 Heart (cardiomyopathy / CHF / arrhythmias) and exocrine glands (e.g. liver cirrhosis) are main organs affected

Causes:
 Transfusional hemochromatosis: pts. getting frequent blood transfusions (e.g. β-thalassemia major); get large
cumulative load, Rx with iron chelation drugs
 Hereditary hemochromatosis: genetic disorder of excessive iron absorption in gut (enterocyte transporter
mutation); Tx with periodic phlebotomy.

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 Folate & B12: Metabolism & Deficiency
Megaloblastic anemias: from reduction in rate of DNA synthesis; RNA/protein synth normal.
 Subset of macrocytic anemias (MCV > 100; abnormally large cells)
 Nuclear-cytoplasmic asynchrony (cytoplasm “matures” faster than nucleus)
can have 2 results: DDx: macrocytic anemia
o cell dies (intermedullary hemolysis / ineffective hematopoesis)  Reticulocytosis (e.g.
o terminal division omitted (big macrocyte formed) hemolytic anemia)
 Liver disease, alcoholism
 Unbalanced growth in all rapidly proliferating cells (bone marrow, tongue
 Drugs
epithelium, small intestine, uterus): look for clinical manifestations here.
 Some myelodysplasias

Pernicious Anemia (B12 defiency) DDx: megaloblastosis:

 Macrocytosis (big RBC) + megaloblastosis (lots of RBC precursors) Interference with DNA synthesis
 Chemotherapeutic drugs
Clinical presentation:  Acute leukemia
Key triad:  Rare inherited disorders
1. Diminished gastric secretions / achylia gastric
(B12 or folate deficiency too!)
2. Megaloblastic anemia
3. Neurologic degeneration (posterior/lateral columns)
Wikipedia on DCH:
Hodgkin's scientific mentor
Patient: older, especially Irish / Scandinavian / English Professor John Desmond Bernal
Signs / Sx: often develops slowly, Asx at first (sometimes neuro abnormalities early) greatly influenced her life both
 Most  least frequent: anemia, paresthesias, GI complaints, glossitis (sore scientifically and politically. He
tongue), difficulty walking was a distinguished scientist of
great repute in the scientific world,
Lab: a member of the Communist
 Macroovalocytes of RBC & hypersegmentation of granulocytes; party… She always referred to him
 Hypercellular bone marrow with lots of erythroid precursors as "Sage" and loved and admired
 Hemolysis: ↑LDH, hyperbilirubinemia, ↑ Fe him unreservedly; intermittently,
they were lovers.

Vitamin B12
Early studies: massive liver feedings helped PA; “extrinsic factor” (B12) in liver & “intrinsic factor” missing in pts
B12: Synthesized by microorganisms; dietary from flesh/milk of ruminant animals
 liver, glandular tissue, muscle, eggs, dairy products, seafood
 Normal body stores 2-5mg, > 1mg stored in liver; daily requirement 2-5 μg (0.1%)
 Significance: B12 stores last at least 1,000 days after absorption stops
Structure: Dorothy Crowfoot Hodgkin
 Porphyrin-like ring with cobalt in center (cobalamin)
 pharm forms are substituted at cyano group & converted by metabolism in vivo

Absorption, Transport, etc.

 Bound to protein in food; released by pepsin (need acid pH) in stomach,
binds to R-substance in gastric juice
 Released from R-substance by trypsin in jejunum
 Intrinsic factor (IF) secreted by gastric parietal cells & complexes with B12
 IF-B12 complex is absorbed EXCLUSIVELY BY TERMINAL ILEUM
(only cells that have IF receptors)
 B12  bloodstream  bound to transcobalamins
(TCII is physiologically relevant)
 TCII receptors in tissues  uptake for use in cell division

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Functions: co-factor for the following reactions
1. Methyl transfer: homocystine + methyl-THF  methionine + THF
o B12 = obligate cofactor for certain folic acid functions
2. Hydrogen transfer: methylmalonyl coA  succinyl coA
o Not involved in folic acid pathway
o High urinary/serum methylmalonyl coA helps distinguish
B12 deficiency from folate deficiency

Clinical findings specific to Vitamin B12 deficiency

1. Low serum B12 levels Schilling test: Pernicious anemia
2. Peripheral / central nervous system disease 1. Phase 1:
a. Classic presentation: “combined system degeneration” a. Give oral radioactive
 dorsal/lat column probs (↓position / vibratory sense), cyanocobalamin + bolus dose
 peripheral neuropathy, unlabeled B12 to block tissue
 cortical abnormalities (“megaloblastic madness”) binding in B12-deficient individuals
3. Methylmalonic acidemia (see above) b. 24h urine collection (need to take
4. Abnormal Schilling test up B12 to get radioactivity into
urine instead of feces).
Causes c. Abnormal (<7% in urine) if IF
Acquired deficiency state: production impaired by Ab (PA),
parietal cells not working (no IF), or
1. Decreased absorption
terminal ileum messed up (no IF-
a. Loss of intrinsic factor receptors)
(gastric atrophy, autoimmune-associated gastric atrophy is #1, also 2. Phase 2: 5 days later
Ab against intrinsic factor / gastrectomy) a. Same process but administer with
b. Terminal ileum disease oral IF; if the defective absorption is
(ileal resection, gluten-induced problems, non-tropical sprue, cancer, corrected, Dx = pernicious anemia
granulomatous lesions, regional enteritis, bacterial overgrowth) (if no history of gastrectomy).
c. Food cobalamin malabsorption
(chronic achlorhydria, proton pump inhibitors, loss of salivary gland function)
2. Inadequate ingestion (vegans / breast-fed infants of vegan moms)

Congenital deficiency state: usually in infancy (failure to thrive, developmental delay, neuro abnormalities, anemia)
 Autosomal-recessive conditions (cobalamin absorption or transport)

Treatment of pernicious anemia: parenteral cyanocobalamin; treat terminal ileum disease or microbes if present

Folic acid
Folate metabolism: we don’t synthesize it; half of body stores are in liver; body stores last about four months
 Most absorbed in proximal jejunum but:
 DIFFUSE DISEASE of INTESTINE is NEEDED for FOLATE MALABSORPTION to occur
 Dietary sources: green leafy veggies, liver, kidney, fruits, mushrooms

Structure / metabolism / function:

 Dietary folate: conjugated with multiple glutamic acids; intestinal deconjugation is needed for absorption
 Needs to be reduced x4 (dihydrofolate reductase) to THF for activity (methotrexate, trimethoprim target)
 Most important function: methyl transfer (e.g. for thymidilate & subsequently DNA synthesis)
 MEGALOBLASTOSIS is from IMPAIRED THYMIDYLATE SYNTHESIS in folate deficiency.

Causes: dietary deficiency is most common (alcoholics, indigents, malnourished)

 ↑ folate requirements with pregnancy / lactation / chronic hemolytic anemia
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  Impaired deconjugation or diffuse intestinal disease can lead to malabsorption
 Blocked utilization (cancer chemotherapy) too Diagnosis of folate defiency
 ↓ serum folic acid (<3ng/mL)
Treatment: 1 mg folic acid PO qd
 ↓ RBC folate levels (<135 ng/mL)
 If caused by methotrexate, coadminister leucovorin (FH4)
 Responds to physiologic doses of folate

Compare & contrast: B12 vs Folate

B12 FOLATE
Megaloblastic anemia Yes Yes
Combined system degeneration Yes No
Dietary Deficiency Rare Common
Dietary Source Muscle, liver, milk, eggs Liver, leafy greens
Deficiency induces hyperhomocysteinemia Yes Yes
Deficiency induces ↑ methylmalonic acid Yes No
Site of absorption Terminal ileum Small bowel
Intrinsic Factor required Yes No

B12, Folate, DNA replication, Neural Tubes, Vascuar Disease, and Cereal

Vitamin B12 deficiency: might trap folic acid as N5-methyl

FH4,
 predominant dietary form (can’t be converted to N-
5,10-methylene FH4 for thymidilate & DNA synthesis)
 Oral folic acid can mostly correct B12 ANEMIA
(possible explanation for why)

Treatment of B12 deficiency with folic acid DOES NOT

CORRECT NEUROLOGIC DEFECTS
 Dietary folate enters @ “folic acid” on top diagram;
via methionine synthase & with B12 participation can
generate methionine (needed for myelin synthesis)
 Pharmacologic folate doesn’t generate methionine,
formate, or SAM (enters at THF stage)
 Cure anemia (can still bump up DNA production) but
hide worsening B12 problem: MUST DDX B12 VS
FOLIC ACID PROBLEM

Folate deficiency & fetal malformations

 Folate deficiency associated with neural tube defects
 0.8 mg folic acid po qd prevents 1st occurrence, 4 mg
prevents neural tube defects in subsequent dose
 USPHS: women in reproductive years should take 400
μg/day of folate supplements

Hyperhomocysteinemia & vascular disease: associated with increased incidence of atherosclerosis / heart disease; folic
acid supplementation reduces homocysteine levels but doesn’t prevent venous/arterial thrombotic disease;
homocysteine may be marker of vascular disease instead of causative agent

Cereal: FDA mandated in 1998 that all cereals be fortified with folic acid to try to bump up folic acid intake; designed for
100μg / day increase (falls well short of USPHS guidelines) efficacy of program not known.
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 Hemolytic Anemia
Hemolytic disorder: any condition where survival time of erythrocyte in circulation < 120 days (normal RBC)

Etiologies:
 Primary (usually congenital): membranopathy, enzymopathy, hemoglobinopathy
 Secondary (usually acquired): immunologic, chemical, physical

Physiologic response (independent of etiology): ↑ rate of delivery of new RBC (↑ reticulocytes)

1. Compensated hemolytic disorder: new increased rate can match destruction
2. Hemolytic anemia: when you can’t compensate (more destruction than new delivery)
a. Max increase is usually 6-8x normal
b. So if RBC life < 15-20 days (1/6-1/8 normal), then hemolytic anemia ensues

Lab techniques
Direct techniques: look at RBC survival time
 Ashby test (historical): transfused mismatched RBCs; follow % cells surviving; problem: not looking @ endogenous RBC)
 Radioactive chromium: most common; binds to hemoglobin (labeling pt’s own RBC)
o Problems: chromium elutes 1%/day from Hb, so have to correct; rate of elution varies with different
Hbs (e.g. faster from SC than normal); has higher affinity for retics, can’t tell blood loss vs hemolysis

Indirect techniques: look at RBC production

 Reticulocyte count (1% circulating RBC normally): increased in hemolytic disorder (abs & %)
o Reticulocyte = cell after most mature nucleated red cell precursor in BM loses nucleus
o Hard to tell in peripheral blood smear: use supravital staining (methylene blue) to clump ribosomes / mitochondria
o Flow cytometry mostly used these days
 Peripheral blood smear:
o young RBC are macrocytes
o polychromatophilia (reticulocytes have diffuse basophilia, look blue when released early from BM)
o nucleated red cells (early release from BM)
 Bone marrow: see more erythroid precursors; usually not required.
 Other: Can see medullary expansion (“hair-on-end” appearance) on radiography; maxillary prominence & expansion of
bones (skull, ribs, hands); sometimes hepatosplenomegaly too (extramedullary hematopoesis & increased sequestration)

Bilirubin
Elevation of indirect bilirubin in hemolytic anemia

Bilirubin review:
1. Breakdown of Hbheme, globin + Fe released
2. Heme  biliverdin (via heme oxidase, CO2 released)
3. Biliverdin  bilirubin
4. Bilirubin bound to albumin when it circulates.
(Unconjugated bilirubin a.k.a. indirect bilirubin =
insoluble, so it doesn’t appear in urine; goes to fat
instead e.g. sclerae)
5. Conjugated bilirubin formed in liver (direct bilirubin,
water-soluble & can be seen in urine)
6. Enterohepatic recirculation from GI to liver or
excretion as urobilinogen from kidneys / stercobilin in
feces

12
Hemolysis: ↑ degradation of Hb  ↑ bilirubin; exceed liver conjugation abilities; see INDIRECT BILIRUBIN in blood
 Conjugated / direct bilirubin doesn’t accumulate (liver can still excrete it)
Liver disease: liver can’t excrete conjugated bilirubin, so DIRECT BILIRUBIN increases in blood

Hemolysis: extravascular & intravascular

Extravascular hemolysis:
 RBC trapped in reticuloendothelial system (liver/spleen/bone
marrow)
 Heme breakdown proceeds through bilirubin pathway; see
increased indirect bilirubin in blood, ↑ CO & Fe

Intravascular hemolysis:
 RBCs destroyed within circulation, Hb released directly into
circulation
 In addition to increased indirect bilirubin, CO & Fe, see
hemoglobin in urine (hemoglobinuria).
 Hb also concentrated by renal tubular cells as hemosiderin,
shed into urine (hemosiderinuria)

Lab stuff

Haptoglobin: α2 globin , binds hemoglobin 1:1, made in liver

 Serum levels vary with age (0 in newborn, higher in older
children & adults); also an acute phase reactive protein
(increased in stress)
 Reduced levels when RBC survival < 90 days
 REDUCED SERUM LEVEL in BOTH intra- & extra-vascular
hemolysis

Hemopexin: β globulin, made in liver, binds heme 1:1

 Serum levels vary with age (higher in older children & adults),
not an acute-phase reactive protein
 REDUCED LEVELS in INTRAVASCULAR hemolysis (mostly)

Methemalbumin: when haptoglobin’s binding capacity exhausted, free Hb combines with albumin → methemalbumin
(means that indirect hemolysis has been liberating Hb into bloodstream)

Carboxyhemoglobin: CO liberated in heme degradation

 rate of CO production directly related to rate of heme degradation (directly related to red cell survival)
 BOTH intra & extra-vascular hemolysis can cause increased levels
 Technically difficult, not used routinely, results messed up in smokers or people with other CO exposure

Summary: distinguishing intravascular & extravascular hemolysis

Both intravascular & extravascular hemolysis Intravascular hemolysis only
 Indirect hyperbilirubinemia  Hemoglobinemia
 ↑ urinary / fecal urobilinogen  Methemalbuminemia
 ↓ haptoglobin / hemoplexin  Hemoglobinuria
 ↑ carboxyhemoglobin  Hemosiderinuria

13
 Classification of Hemolytic Disorders: intracorpuscular vs extracorpuscular defects

Intracorpuscular defect: etiologic factor intrinsic to red cell; most often genetic
 Membranopathy: see elliptocytes & spherocytes
 Hemoglobinopathy: e.g. sickle cell trait / dz
 Enzymopathy: e.g. pyruvate kinase deficiency

Extracorpuscular defect: etiologic factor extrinsic to red cell; most often acquired
 Thermal injury, mechanical injury (e.g. Waring blender syndrome from heart valve, etc.), toxic injury,
 Antibodies (autoimmune hemolytic anemia)

Intra & extra-corpuscular defects combined too: drugs, for example

Mophologic abnormalities
Hereditary Spherocytosis: intracorpuscular membranopathy

 Membranopathy: disturbance in protein-protein interaction (within RBC cytoskeleton or links to cell membrane)
 Spectrin abnormality (or spectrin binding proteins): cytoskeletal proteins involved in vertical (cytoskeleton –
PM) and horizontal (cytoskeleton – cytoskeleton) interactions

 Unstable red cell membrane loss of membrane  SA reduced  sphere forms (smallest SA)

o Cells rupture more easily in hypotonic solutions

(osmotic fragility test)

o Direct relationship between degree of spectrin

deficiency and: osmotic fragility, reticulocytosis,
depression of haptoglobin, severity of anemia

Autoimmune Hemolytic Anemia: extracorpuscular defect

 Ab coat RBC; as RES removes Ab, bits of

membrane get removed: reduction of surface
area  sphering
 Detection: Coomb’s test

DIRECT COOMB’S TEST INDIRECT COOMB’S TEST

Detect antibody attached Detect anti-RBC antibody in
to patient’s RBC surface patient’s serum
Use agglutinating Expose control RBCs to patient
antibody or labeled anti- serum; look for Ab binding to
human IgG control RBCs’ surface
 Can be positive in autoimmune
hemolytic anemia - if enough
Ab produced to exceed capacity
Positive for autoimmune of RBC to bind it
hemolytic anemia  Positive if antibodies are made
against foreign RBC antigens
(post-transfusion, feto-maternal
incompatibility)

14
G-6-PD deficiency: combined extra- and intracorpuscular defect

 Cells sensitive to oxidants: either an absence or dysfunction of glucose-6-phosphate dehydrogenase

 Most common form: A- variant; enzyme functions but has a shorter half-life
o younger RBC have a higher level of G6PD
o If you induce oxidative stress (primaquine), anemia, reticulocytosis,
hemoglobinuria all go away
o Reticulocytosis pumps out more young RBC, so G6PD levels on average are
better. See picture (Hb on top, retics on bottom, primaquine given at t=0)

When hemoglobin falls & red cells start to lyse (after an oxidative stress trigger):
 Heinz bodies (intracellular precipitates of hemoglobin)
 Dark urine (hemoglobinuria/methemalbuminuria)
 Jaundice

Pathophysiology of G-6-PD Deficiency

G-6-PD: catalyzes conversion of G6P to 6PG, reducing NADP to NADPH
G-6-PD deficiency: Cells can’t provide enough reduced glutathione following oxidative stress
 (can’t protect cellular elements against oxidation  symptoms)

Details of pathophys / metabolism:

 G6P usually metabolized mainly through glycolysis, small proportion
through pentose phosphate pathway

 Normal patient:
o Oxidative stress situation: increase in conversion of NADP to
NADPH (more G6PD activity, more 6PG produced, more
glucose utilization, and more pentose shunt activity)
o H2O2 & free radicals oxidize GSH, forming mixed disulfides of
Hb and glutathione
o NADPH is used to return glutathione to reduced state & remove peroxides

 G6PD Deficiency: can’t increase flow through PPP when oxidative stress occurs
1. NADPH can’t be generated as in the normal patient
2. Can’t regenerate GSH (glutathione  GSH conversion needs NADPH)
3. Hb starts to precipitate (Heinz bodies); membrane lipids get peroxidated  red cells destroyed

15
 Hemoglobinopathies
Hemoglobin chains & forms
Genes
Chromosome Genes HEMOGLOBIN TYPES: QUICK GUIDE
β-globin cluster 11 Beta(β), Delta (δ), Gamma (γ), Epsilon (ϵ) Hb A (“adult hemoglobin”) α2β2
α-globin cluster 16 Alpha (α), Zeta (ζ) Hb A2 (“minor adult”) α2δ2
Hb F (“fetal hemoglobin”) α2γ2
To make a hemoglobin: pick one from each cluster HbE (“embryonic hemoglobin”) α2ϵ2
Produced only just after conception ζ2ϵ2
Changes through the lifespan:
Erythropoesis happens in different organs throughout embryonic development (sequential):
 yolk sac  liver/spleen  bone marrow

Hb expression
 Just after conception: ζ and ϵ chains expressed
(ζ2ϵ2 predominates)
 Major switch #1: embryonic ζ chains replaced by
adult α chains
(shortly after conception, α2ϵ2 predominates)
o From this point on, it’s all α chains and no more ζ
o Embryonic ϵ replaced by fetal γ shortly thereafter
(α2γ2 predominates)
 Major switch #2: fetal γ chains replaced by adult β
chains (α2β2 predominates)
o This change is INCOMPLETE and REVERSIBLE
(basis for some therapy)
 Adult hemoglobins: 95% Hb A (α2β2), also 2% Hb A2 (α2δ2) and 2% Hb F (α2γ2)

The locus control region (chromosome 11) along with part of the 5’ β-globin complex help modulate this switching
 Transacting factors bind to LCR; LCR loops over & silences globin genes’ promoters (physical DNA rearrangement)
 Mutations can mess up expression patterns; gene therapy (FDA-approved!) and other therapies can take advantage

Hemoglobinopathies: overview

Hemoglobinopathy: mutation of either the alpha or beta globin genes which leads to:
 Varient globin molecule, decreased globin production, or absence of globin production
 Every mutation type you can think of has been described in globin genes, including regulatory / noncoding areas

Five clinical syndromes:

 Sickle syndromes (sickle cell trait, SC disease, etc)  hemolytic anemia, vaso-occlusive events (pain / infarction)
 Unstable hemoglobins  drug induced hemolytic anemia
 Oxygen affinity variations / M hemoglobin  cyanosis / polycythemia
 Thalassemia (α / β types, microcytic anemia + iron load from transfusion dependence, from α / β imbalance)

16
 Sickle Cell Anemia

Hb S: a different type of Hb (like Hb A, F, etc.) where there’s a β6 GLU to VAL mutation

HB C: a different type of Hb too (B6 LYS to VAL); doesn’t form polymers as readily as S (but more readily than A)
Remember that Hb A is normal adult Hb

Genotype Phenotype FEATURES of SICKLE CELL ANEMIA

AA Normal  Hemolytic anemia (shortened red cell survival)
SS Sickle cell disease  Complex pathophysiology  variable severity
AS Sickle cell trait: no sickling in vivo; same survival as AA  “Gene-switching” therapy
SC SC disease (compound heterozygote); less sx than SS

Pathophysiology
Polymer formation: β6 Glu  Val mutation fits into a
hydrophobic pocket in adjacent β chain of another tetramer
 Other interactions are favorable & stabilize too
 Hb in RBC is really concentrated (97-8% protein is
Hb!) → these polymers are big deal!

Polymers & crisis:

1. Polymer formation is concentration
dependent: in normal situations,
polymers are in reversible equilibrium
with monomers
2. After you hit a certain point (“critical
polymer”), growth of the polymer is thermodynamically favorable
3. Polymers  rigidity, changes in cell shape, membrane distortion
(SICKLING!) SC crisis:
 Sudden in onset
4. Classic theory: Rigid RBC  obstruction of blood flow  tissue  Unpredictable (and 80% SS have < 1 crisis/yr)
hypoxia  tissue damage (localized pain + swelling); repeated low  Variable: even among individuals with
grade obstruction  organ damage (spleen, lungs infarct) identical sickle mutations!
 Multifocal
5. More current ideas: COMPLEX, MULTIPLE EVENTS (RBC, WBC, Suggests that classical theory is incomplete
coagulation)
o RBCs block bifurcations in blood vessels, pre-capillary arterioles (why crises manifest in multiple places!)
o Membrane distortion of RBC  expose new proteins  stick to endothelium (along with RBC)
o NITRIC OXIDE: Intravascular hemolysis, arginase leakage,  ↓NO  inflammation & vasoconstriction
(NO levels would explain some of the systemic nature of the crisis)
o Coagulation cascade activated too

6. Genetic modifiers are key too: not simply a single gene

o WBC count, Hb F levels, cytokine production, endothelial surface proteins, even in opiate receptors
(some patients don’t respond as strongly; docs think they’re malingering!)

Hb F and Sickle Cell Disease

 Increased Hb F corresponds to less pain (Hb F doesn’t form polymers)
 Newborns therefore have no symptoms even if SS (enough Hb F around to inhibit Hb S polymerization)
 Hydroxyurea (and two other drugs): can increase Hb F in SS patients (4% 15%, 50% reduction in pain crises,
hospitalizations, mortality).
 Other treatment: transfusions, bone marrow transplant if needed, others
17
 Lab tests for sickle cell
(know how these compliment one another for the exam)

Sickle Prep (Sickledex™, sickle solubility test)

1. Put drop of blood in sodium hydrosulfite solution
2. RBC lyses, releases Hb
o Hb A goes into solution (clear)
o Hb S precipitates (insoluble  cloudy)
3. ALL THREE TYPES (SS, AS, SC) ARE POSITIVE (just screening for presence of Hb S) – CAN’T DISTINGUISH
Sickle Prep Hb electrophoresis
Hb Electrophoresis
Speed Fast Slow
1. Run sample on electrophoresis; measure migration
Can distinguish
2. CAN DISTINGUISH SS vs SC vs AS vs AA Nope Yep
AA/AS/SS/SC

Thalassemia
DISEASE OF GLOBIN CHAIN IMBALANCE
Mediterranean populations (Italians, Greeks, Arabs, Africans) & Southeast Asia α>β Beta thalassemia
β>α Alpha thalassemia
Normal ratio β:alpha = 1.0 (beta thal <1, alpha thal > 1)

Beta thalassemia
 100+ mutations, decrease in β globin production, don’t alter β-globin protein (normal Hb A, just less)
 Excess alpha chains  unstable α4 tetramers  precipitate  RBC damage  hemolysis
o γ chains are still around: make α2γ2 (Hb F) in increased levels
o δ chains are around in adults: make α2δ2 (Hb A2) in increased levels too
o Hb electrophoresis: see increase in Hb F and Hb A2 relative to Hb A

Presentations:
Genotype Name Features
Heterozygote Thalassemia minor, thalassemia trait Small RBCs, minimal anemia
Homozygote Thalassemia major Transfusion-dependent (severe anemia)
(“Cooley’s anemia”) Microcytic / hemolytic anemia

Compound heterozygote Thalassemia intermedia Thal major with 5+ alpha genes = more mild
Thal minor with 4- alpha genes = more severe

Diagnosis:
 microcytic anemia (decreased Hb, low MCV, hypochromic, etc.)
 increased RBC number (marrow trying to compensate  nucleated RBC in periphery)
 Elevated Hb F and Hb A2 relative to Hb A
 Features of chronic anemia (thal major): Hepatosplenomegaly (extramedullary hematopoesis), brittle bones /
enlarged marrow space (hypertrophy of skull/facial bones, hair on end appearance)

Therapy:
 prenatal dx (incidence has declined in countries where risk was high)
 hypertransfusion + iron chelation (would overload otherwise; no excretion mechanism)
 bone marrow transplant if severe; Hb F “switching agents” (e.g. hydroxyurea) might not make enough Hb F to help

18
Alpha thalassemia
 Excess beta chains → unstable β4 tetramers  precipitate  RBC damage  hemolysis
 In newborn: γ chains make γ4 tetramers (no alphas around)  “Hb Bart’s”; can detect it but useless
o δ chains don’t do you any good either, since you’re low on α chains! Nothing to pair it with.
o Hb electrophoresis: Hb F, Hb A2 don’t change relative to Hb A (all need α chains, so all decrease!)

Common in Black, Italian, Greek, Arab, Asian, Indonesian populations

 Tremendous selection factor (See map)

Genotype Phenotype Features

4 alpha genes Normal Normal
3 alpha genes “Silent carrier” No Sx but can pass on gene
2 alpha genes Trait Microcytic anemia (like β thalassemia trait &
mild iron deficiency anemia in severity)
1 alpha gene Hb H Severe hemolytic anemia
0 alpha genes Hydrops fetalis Inconsistent with life (die in utero)

Diagnosis:
 Microcytic RBC ± anemia
 Often confused with iron deficiency
 Hb A2 / Hb F levels normal relative to Hb A so Hb electrophoresis not helpful!
 RDW / MCHC not reliable; family studies may not be helpful
 Rely on clinical history (race of patient); must rule out iron deficiency anemia as microcytic anemia cause
o E.g. put ‘em on Fe and they don’t get better! Think α-thal!

Methemoglobin (Fe3+): a Hb variant

Doesn’t lead to anemia; can cause decreased oxygen transport  (pseudo)cyanosis

 Blood color (Hb color) altered: RED (Fe+2)  BROWN (Fe+3)
 Can confuse with: pulmonary or cardiovascular disease

Causes: genetic or acquired

 Aut-dom: congenital variants in alpha or beta chain that stabilize heme molecule in Fe+3 (ferric) state
 Aut-rec: deficiency of enzymes in RBC that help keep heme molecules in Fe+2 state (Reduced)
 Acquried: oxidant damage of hemoglobin (drugs / toxins – especially nitrates)
o Too much nitric oxide (used to close PDA in infant)
o Diarrhea (nitrates overproduced by bacteria)
o Poisoning (nitrates from fertilizer in well water)
o Other drugs: nitroglycerin, sulfonamides, napthaline, others with nitrates.

19
 Hemostasis
Background: blood under pressure; breaks in vascular continuity  exsanguination (need to seal it off)

 Procoagulants: Platelets & coagulation proteins work together to stop bleeding

1. Vessel walls contract (reduce vascular flow  ↓bleeding)
2. Platelets adhere (vWF, etc.), provide phospholipid-rich environment for coagulation factors
3. Cross-linked fibrin network (via coagulation cascade)

 Anticoagulants: Balanced against procoagulants (

1. Endogenous anti-thrombotic proteins (dampen procoagulant coag cascade)
2. Fibrinolytic system (remodel / dissolve clots)

Hemostasis

General principles: basic goal is to get to formation & cross-linking of fibrin

 Extrinsic pathway more important in initiation

o Requires “extrinsic” tissue factor
o Produces some thrombin (IIa)
o Tissue factor inhibitor shuts it down pretty quickly
(released by intact epithelium’s endothelial cells)

 Intrinsic pathway big in amplification / propogation

o (heats up after thrombin production by extrinsic pathway)

20
 The Coagulation Cascade

XII Intrinsic Extrinsic

Pathway
Surface kallikrein Pathway
HMW kininogen

IX
XIIa TF + VII
XI XIa Ca++
XI Ca++ Phospholipid

X
Ca++
IXa Phospholipid Ca++
IIa TF + VIIa
VIII VIIIa

Xa

V IIa Va
V fibrinogen

II IIa
Ca++
Phospholipid

fibrin monomer

IIa
XIII XIIIa

fibrin clot

THE PLAYERS
Serine proteases XII, XI, X, IX, VII, II (prothrombin), prekallikrein Circulate in blood as zymogens, cleave & activate others
Cofactors  VIII (cofactor for IX) Increase reaction kinetics (1000 fold) by localizing /
 V (cofactor for X) concentrating partners to membrane surfaces (optimize
 HMWK* ( cofactor for XI & prekallikrein) reaction)
 Tissue factor (cofactor for VII)
Glycoprotein Fibrinogen Becomes insoluble (fibrin) after thrombin cleaves it
Transglutaminase XIII  Cross-links fibrin strands covalently
 Links α2-antiplasmin to fibrin |
(inhibits plasmin, part of fibrinolytic system)
 If no XII: clots fall apart
Vitamin-K-  II, VII, IX, X  Need vitamin K for synthesis (warfarin inhibits)
dependent  Proteins C, S  Vit K needed for addition of γ-carboxy glutamic acid side
chains (allow proteins to bind phospholipid-rich
membranes in presence of calcium
 No Vit K  no side chains  no binding, less activity
*HMWK = high-molecular-weight kininogen
21
 Steps of Coagulation Cascade (in more detail)
EXTRINSIC PATHWAY
Requirements Anticoagulant Associated Disease
Step Description
Ca PLs Vit K Target? State?
0 Factor VII normally circulates; small amounts of VIIa also in blood (VII needs vitamin K)
1 Vascular damage  Tissue factor exposed in subendothelium (normally hidden)
2 Factor VIIa forms complex with tissue factor
3 VIIa + TF cleaves X  Xa (X requires vitamin K)
INTRINSIC PATHWAY
Requirements Anticoagulant Associated Disease
Step Description
Ca+2 PLs Vit K Target? State?
Factor XII activated by exposure to highly negatively charged surfaces (e.g. endothelium)
0
Note: Not important in vivo (just for assays)
XIIa activates prekallikrein  kallikrien; high molecular weight kininogen is a cofactor
1
(concentrates prekallikrein on the membrane where XIIa generated)
1.5 Kallikrein activates more XII  XIIa (positive feedback)
2 XIIa also activates Factor XI (HMWK is cofactor, concentrates XI on membrane)
XIa activates Factor IX  IXa Hemophilia B:
3 ++ ATIII (XIa  XIi)
(needs Ca and phospholipids – surface of activated platelets; IXa needs Vit K for synth) Factor IX deficiency
Hemophilia A:
Proteins C/S
3.5 Meanwhile, VIII activated by thrombin (Factor IIa)  VIIIa Factor VIII deficiency
(VIIIaVIIIi)
Von Willebrand Disease
+2
4 VIIIa + IXa form “tenase” complex, assembles on PL-rich platelets using Ca
VIIIa+IXa (tenase) cleaves X  Xa
5 +2 ATIII (IXa  IXi)
(X also requires vit K, binds to PL using Ca using γ-carboxy-glutamic acid side chains)
COMMON PATHWAY
Requirements Anticoagulant Associated Disease
Step Description
Ca+2 PLs Vit K Target? State?
XXa either by VIIa/tissue-factor (extrinsic) or IXa/VIIIa (intrinsic)
0
(X needs vitamin K, happens on platelet surfaces with Ca + PLs)
Factor V Leiden
1 V  Va via thrombin (IIa) activation Prot. C/S (VaVi)
(hypercoaguable)
Va + Xa (prothrombinase complex) cleaves prothrombin (II)  thrombin (IIa) Thrombin deficiency =
2 ATIII (Xa  Xi)
(thrombin needs Vit K for synthesis) embryonic lethal
3 Thrombin (IIa) cleaves fibrinogen  fibrin monomers ATIII (IIa  IIi)
4 Fibrin monomers polymerize (loose fibrin clot, hydrostatic bonds
5 Thrombin activates XIII  XIIIa
4 XIII is a transglutaminase; catalyzes fibrin cross-linking & affixes α2-antiplasmin
22
Observations:
 When thrombin gets cleaved, things really start rolling: more cofactors! VIIIVIIIa (intrinsic), VVa (common)
o Thrombin also activates platelets and protein C (anti-thrombotic protein!)
 When vitamin-K-activated stuff is involved (II, VII, IX, X) the action needs calcium and takes place on
phospholipids of platelet surfaces (that’s where γ-carboxy glutamic acid side chains like to do their thing)

Lab Tests
Basic idea for both PT & APTT:
1. draw blood into sodium citrate sol’n (chelate calcium)
2. centrifuge (keep plasma only)
3. add phospholipids (platelets were removed) and calcium
4. add XII for APTT (intrinsic pathway) or tissue factor for PT (extrinsic pathway)
5. Measure time to clot formation based on light absorption

Prothrombin Time (PT) Activated Partial Thromboplastin Time (APTT, PTT)

Extrinsic Pathway YES (VII) no
Intrinsic Pathway no YES (XII, HMWK, prekallikrein, XI, VII)
Common Pathway YES (X, V, II, fibrinogen)
Factor XIII NEITHER (need test of clot strength; these are just for clot formation)

Mixing test:
 Prolongation of PT or APTT can be from either deficit in coagulation factor or Ab against coagulation factor
 Mix 1:1 patient:normal plasma, re-run prolonged test.
o If it corrects, it was deficiency (50% of factor sufficient to normalize test)
o If it’s still prolonged, abnormality is from antibodies (neutralizing factors in normal plasma too)

Factor levels
 Isolate a specific factor that’s the issue: use factor deficient plasma (all but one factor) and add patient plasma
o Run test (APTT or PT depending on pathway you suspect) – will be dependent on patient’s factor level
o set up standard curve with length of test vs % of factor (controls), determine patient’s level

Diseases
Hemophilia A: congenital factor VIII deficiency
Treatment of hemophilia A:
 X-linked, recessive (1/10k males)
 Recombinant/plasma-derived
 Deep tissue bleeds, hemarthroses (joint bleeds)
factor VIII
 Severity varies with factor VIII level (mild > 5%, moderate 1-5, severe >1%)
 DDAVP (desmopressin) to
release VIII & vWF (mild only)
Hemophilia B: congenital Factor IX deficiency
 X-linked inheritance (1/50k males)
Treatment of hemophilia B:
 Similar manifestations to hemophilia A
 Factor IX concentrates
Von Willebrand disease: congenital vWF deficiency
Treatment of vWD:
 Autosomal, most common inherited bleeding disorder (1 in 1000)
 mild vWD  DDAVP (makes
 less vWF, which is a carrier protein for VIII in the bloodstream (protects
endothelial cells release vWF);
from C/S inactivation); ); less severe than hemophilia A
 severe  factor VIII
 More important: ↓adhesion of platelets to subendothelial collagen
concentrates (have vWF)
 Bleeding from mucosal surfaces rather than deep tissue bleeds (platelets!)
 Tests:
o ELISA (vWF antigen test) used to measure vWF levels
o Ristocetin Cofactor Assay (antibiotic; causes vWF-dependent platelet aggregation, tests vWF function
23
Vitamin K deficiency: MOST COMMON ACQUIRED BLEEDING DISORDER
Etiologies of Vit K deficiency:
 Immature forms of II (prothrombin), VII, IX, X
 broad-spectrum abx
o Also C and S
 surgery
 Half-lives vary: VII has shortest half-life  prolonged PT 1st (extrinsic)
 poor nutrition
o Eventually PT & APTT both abnormal
 excessive biliary drainage
 Treatment: Vitamin K (oral, sub-q, IV)
 warfarin

Liver disease: all coagulation proteins made in the liver

 Severe liver disease only: need ~90% loss of function before PT/APTT slowed; means poor prognosis
 Factor VIII synthesized outside of hepatocyte; not affected
 Treatment: plasma

24
 Thrombosis
Balance between procoagulant & anticoagulant forces; imbalance  hemorrhage or thrombosis

Circulation & Endothelial Cells: Anticoagulants Procoagulant:

Circulation: prevent local accumulation of activated coagulation factors & incidental  platelets
thrombous formation  coagulation factors
Antithrombotic:
Endothelial cells:  circulation
 express thrombomodulin (binds thrombin, activates protein C)  endothelial cells
 don’t express tissue factor  fibrinolytic system
 endogenous anti-
 produce prostacyclin (PGI2): inhibits platelet function
coagulant proteins
 promote fibrinolysis (tissue plasminogen activator: activates plasminogen)

Endogenous anticoagulant proteins

Antithrombin III: inactivates IIa, IXa, Xa, XIa
 1st to be discovered; synthesized in liver
 Serine protease inhibitor: suicide substrate for IIa, IXa, Xa, and XIa
 Inhibitory action increased 1000-fold if heparin is around (used to treat thrombosis)

Proteins C & S: inactivate Factor Va & VIIIa

Both vitamin K-dependent; bind PL-rich surfaces like activated platelets when calcium’s around

Protein C: serine protease; Protein C  activated protein C (APC)

 Activated by thrombin when bound to thrombomodulin (endothelial cells)

Protein S: in plasma; free (60%) and bound to C4b binding protein (40%)
 Need free form to participate as cofactor for protein C
 C4b binding protein increased in pregnancy & estrogens / OCP (bind more protein S, hypercoagulable)

Activity: APC + free protein S make complex; inactivate Factor Va (Xa cofactor) and factor VIIIa (IXa cofactor)

Fibrinolytic system
Purpose: lyse fibrin clots

Plasminogen: major fibinolytic enzyme

 circulates in inactive form in plasma, activated by two different enzymes (plasminogen  plasmin)
o Tissue plasminogen activator: from endothelial cells; mostly activates plasminogen on clot surface
o Urokinase can activate plasminogenplasmin too
o Plasminogen activator inhibitor I opposes this process (inhibits TPA / UK)
 TPA / UK can be given for thrombotic disease (MI, PE) TPA UK

pharmacologically Plasminogen Plasmin

Plasmin: cleaves fibrin into fragments (fibrin degradation products, FDP) Plasminogen activator inhibitor I

 Opposed by α2-antiplasmin (connected to clot surface by Factor

XIIIa); prevents premature clot lysis Fibrin FDP
2-antiplasmin

25
 Disease states

Pathological venous thrombosis: imbalance between prothrombotic/antithrombotic mechanisms; congenital/acquired

 Usually need several risk factors
(risk adds up, then you go over the top & clot) Acquired risk factors for venous thrombosis
 Immobility
(↓ clearance of coagulation factors)
Factor V Leiden: congenital risk factor for venous thromb  Estrogens / OCP
 Mutation (ARG 506 GLU) in the spot where APC (↑ synthesis of clotting factors VIII, vWF, fibrinogen;
cleaves V to inactivate it ↑ c4b binding protein  ↓ free protein S)
 End result: Factor V resistant to APC (↑ clotting)  Surgery/trauma
 Older age (↑ coagulation factor levels)
Epidemiology: 5% US Caucasians, up to 15% Swedish are  Pregnancy (like estrogen therapy)
heterozygous; uncommon in other groups.  Cancer (hypercoagulable; ↑tissue factor production?)
 Heterozygotes: 5x higher risk;
Homozygotes: 50x higher risk of venous thromboembolism
 Risk amplified if other risk factors present (heterozygote + OCP  30X RR of venous thrombosis)
 Does not increase risk of arterial thrombotic events

Lab tests:
 Activated protein C resistance assay (functional test)
time
o Add APC to patient plasma diluted 1:5 in factor V deficient plasma, do APTT, calculate timewithout APC
with APC
o Normal pt: adding APC prolongs APTT by > 2.2 fold
o Factor V Leiden: less of an APC effect (1.6-fold longer for heterozygotes, >1.3-fold for homozygotes)
 PCR-based assay: used to confirm genetically

Treatment: only if symptomatic (use anticoagulants)

Prothrombin 20210: gene mutation, newly acquired risk factor for venous thrombosis
 3’UTR of prothrombin  increased translation efficiency  ↑ prothrombin levels (25% higher)
 Heterozygotes: 2% Caucasians; uncommon in other groups; 2-3x increased risk
 Diagnosis: molecular biology; Treatment only if symptomatic

Deficiency of Antithrombin III: example of a deficiency in an anticoagulant protein, predisposes to venous thromb.
 ATIII inactivates IIa, IXa, Xa, IXa
 Heterozygotes: 1/5k; homozygotes  embryonic death
 20x increased risk of thrombosis! (heterozygotes! One of most potent inherited prothrombic states)
 Can also acquire (liver dz, nephrotic syndrome – don’t make as much or spill it into urine)
 Dx: measure activity in plasma samples

Protein C/S deficiency: another example of a deficiency in an anticoagulant protein, predisposes to venous thromb.
 5-10x risk of venous thrombosis; Diagnosis: measure activity in patient samples

Protein C deficiency: 1/250-500 heterozygotes; lower # with sx

 Homozygous is rare; causes diffuse neonatal thrombosis (purpura fulminans)
 Acquired from vitamin K deficiency / warfarin; liver disease

Protein S deficiency: 1/1000 heterozygotes

 Can be acquired like protein C; also pregnancy/estrogen/OCP (C4b binding protein levels)
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Defects in fibrinolytic system not identified so far as venous thrombosis risk factor

Antiphospholipid Antibody Syndrome: predisposes to BOTH ARTERIAL AND VENOUS thrombosis

 Ab against phospholipid binding proteins (e.g. beta-2-glycoprotein I; binds to endothelial-bound phospholipids)
 Ab binding triggers tissue factor expression by endothelial cells; C’ activated, endothelial damage
o End result: coagulation via extrinsic pathway  thrombosis

Signs / sx:
 Fetal loss (placental thrombosis)
 Thrombocytopenia (activation/consumption of platelets; immunological destruction

More common in pts with other autoimmune disorders (SLE, RA) but also viral infections (HIV) or cancers

Diagnosis: very important

Important: These pts have higher risk for recurrent thrombotic events; require longer durations of anticoagulant Tx

1. ELISA (phospholipid antigen substrate, add patient sample, detect with anti-human IgG/M/A)

2. Coagulation assays: sensitive to antiphospholipid Ab, which cause prolongation of clotting in vitro
 APTT: use reagents with low phospholipid content, look for slowing
o Mixing studies to follow up (won’t correct; Ab will still neutralize PLs)

 Dilute Russell viper venom time (dRVVT)

o Directly activates factor X in common pathway (DIC after a snake bite!)
o Reaction & subsequent ones are phospholipid dependent; sensitive to anti-PL Abs
o Anything reducing X, V, prothrombin, fibrinogen would also slow test
 Mixing studies to follow up (won’t correct if inhibitor like antiphospholipid Ab present but will if
due to warfarin, vitamin K deficiency, etc. – normal pt plasma will have enough X, V, etc around)
 Add purified phospholipids and repeat dRVVT: will correct (tons of PLs, bind all the antibodies,
still have PLs around for reaction to take place)

Hyperhomocysteinemia
 Skipped over mostly in lecture; importance diminishing in
recent years
 Acquired/congenital cause for both arterial & venous
thrombosis
 Deficiencies of folate / B12 / B5 result in
hyperhomocysteinemia; mutations too
 Risk increased for venous thrombosis and myocardial
infarction
 High homocysteine  tissue factor expression +
endothelial damage; renal failure can result
 need folate / B12 supplementation

Disseminated Intravascular Coagulation (DIC)

 Acquired coagulation disorder DIC: Associated with…
 Trauma
Pathophysiology
 Sepsis
 excessive activation of clotting cascade  widespread microvascular thrombosis
 Snakebites
 consumption of clotting factors, platelets, endogenous anticoagulant
 Tumors
proteins & activation of fibrinolytic system bleeding
 Amniotic fluid emboli
 Common start: release of activators of coagulation (tissue factor / thrombin /
other activated serine proteases)

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Plasmin working like crazy (fibrinolytic system)
 digests fibrin clots into D-dimers (fragments)
 Can use ELISA to detect D-dimers in plasma (good in diagnosis)

Tx by detecting & correcting cause

DIC: Levels
 ↑D-dimers (plasmin!)
 ↑APTT/PT (used up all your clotting stuff!)
 ↓platelets
 ↓fibrinogen

Hemostasis: the whole big, ugly picture

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