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


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) Most primitive Myeloid (“ high quality”) (“low quality”) All lympho-hematopoetic Granulocytes, RBCs, lineages platelets, B-cells(?) Rarely involved Most “stem cell disorders” Delayed but life-long Rapid but limited CD34+/-, other markers CD34+, other markers +, mostly -, smaller larger +++ + (or low)

OTHER PLAYERS (IN STROMA) Growth factor/cytokine Stimulates…
Erythropoietin (kidneys) Thrombopoietin (liver) Flt-3 Stem cell factor G-CSF RBC Platelets, HSC Dendritic cells, HSC Mast cells, HSC PMNs

Precursor to: Disease Engraftment Phenotype Aldehyde DH

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)

      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 o o White colonies on assay: makes white blood cells Red colonies on assay; making RBCs Mixed coloration on assay; making WBC & RBC too

BFU-E: burst-forming unit erythroid: RBC progenitor CFU-Mixed / CFU-GEMM: progenitor of both CFU-GM and BFU-E (probably like CFU-S)

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


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 (HIV, parvovirus) apoptosis Called a “myeloproliferative” disorder – is really a “ myelo-accumulative disorder”

4. Viruses

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


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 (ability to unload oxygen) hyperdynamic precordium, tinnitus/roaring in ears 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

2,3 DPG pH Oxygen Temperature

Right Left Left Right

↓Decreases ↑Increases ↑Increases ↓Decreases

You’re bumping O2 off to deliver more to tissues (which is 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 oxygen. Blood gets more acidic from CO2, pH drops, oxygen affinity decreases, and more O2 gets dropped off 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 much O2


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

Clinical Tests & Definitions
QUESTION Is the patient anemic? RBC production/destruction/loss? Micro/macro/normocytic Hypo/normochromic Morphology Hct =
PCV Vblood

TEST CBC, Hb, Hct Reticulocyte count (usually ~1%) RBC Indices Peripheral blood smear Hb =
g Hb dL blood

; PCV = packed cell volume (packed RBC volume)
Hct RBC Count Hb

Mean Corpuscular Volume: Mean Cell Hemoglobin:

reflects average size / volume of RBC (in fl, femoliters) reflects weight of Hb in average red cell indicates concentration of Hb in average red cell (%)

MCH = RBC Count
Hb Hct

Mean Cell Hemoglobin Concentration: MCHC =

Reticulocyte = young RBC Normal morphology: donut shape, center pallor 1/3 of red cell

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


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)
lumen mucosal cell blood

From food to blood: (remember that Fe is very oxidative / dangerous & body needs protection from it) 1. Absorption: brush border of upper small intestine via transport proteins 2. Transport: Binds to apotransferrin in mucosal cell  forms transferrin exported to blood (intracellular apotransferrin recycled)  exported to blood  bound to soluble transferrin in blood 3. Uptake: cells that need iron have transferrin receptors, e.g. erythroid precursors 4. Storage: mostly in Mϕ of reticuloendithelial system (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






di FeTf ApoTf TfR mono FeTf

Pathophysiology of Iron Deficiency
1. 2. 3. 4. 5. Iron stores depleted Fe becomes limiting factor in heme biosynthesis ↓ heme  ↓hemoglobin assembly ↓Hb  ↓ RBC production 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) 6

Iron sufficient state:

Iron deficiency: Iron response proteins (IRP-1 & 2) o IRP-1, IRP-2 lose their iron atoms (↓iron around); start RNAbinding (bind iron response element: IRE) o Bind IRE in transferrin mRNA & stabilize  ↑transferrin production (↑iron transport) o Bind IREs in ferritin / ALA synthase mRNAs & block translation start site o IRP-1 is aconitase (cytoplasmic TCA cycle enzyme); loses enzymatic activity if no iron (increasing iron for it’s own use!)
Iron sufficient state TCA enzyme activity mRNA unstable ↓ transport mRNA transcribed ↑ storage mRNA transcribed ↑ heme synthesis Iron deficient state IRP-1 functions IRP stabilizes mRNA ↑ transport IRP blocks transcription ↓storage IRP blocks transcription ↓heme synthesis




transferrin ferritin heme



Iron deficient state:

enzyme inactive; becomes IRP

transferrin ferritin heme


Aconitase Transferritin Ferritin ALA synthase



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) General symptoms (all anemias)  Pallor  Fatigability, weakness  Dizziness  Irritability Causes:  recurrent / chronic / occult blood lost (e.g. GI bleed)  failure to meet physiological requirements (rapid growth, menses /
 pregnancy / lactation) Inadequate intake (diet low in heme iron, e.g. strict vegans; GI disease, surgery, excessive milk intake in infants) pagophagia pica glossitis angular stomatitis koilonychia blue sclerae


craving ice craving of nonfood substances (dirt, clay, laundry starch) smooth tongue cracking of corners of mouth thin, brittle, spoon-shaped fingernails

Lab findings

Peripheral smear:  Microcytic & hypochromic  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) 7

Sequential changes in lab values 1. Ferritin decreases: stores being depleted 2. Iron saturation (iron/transferrin) decreases: you’re iron deficient! 3. MCV, Hb, Hct decrease: anemia! Hb production is limited now Generally, microcytosis develops before significant anemia

Response to therapy Peak reticulocyte count 7 - 10 days Increased Hb and Hct 14 - 21 days Normal Hb and Hct 2 months 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 DDx vs IDA can be difficult:
   ferritin increases in ACD (want to store more iron) total iron binding capacity ( transferrin) increases in IDA (transport more) Gets even crazier if both present at once

Iron deficiency vs. Anemia of Inflammation IDA Ferritin Serum Iron TIBC sTfR Marrow Iron No  No  ? Inflammation Both ?

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.


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) DDx: macrocytic anemia can have 2 results:  Reticulocytosis (e.g. o cell dies (intermedullary hemolysis / ineffective hematopoesis) hemolytic anemia) o terminal division omitted (big macrocyte formed)  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)
 Macrocytosis (big RBC) + megaloblastosis (lots of RBC precursors) Clinical presentation: Key triad: 1. Diminished gastric secretions / achylia gastric 2. Megaloblastic anemia 3. Neurologic degeneration (posterior/lateral columns) Patient: older, especially Irish / Scandinavian / English Signs / Sx: often develops slowly, Asx at first (sometimes neuro abnormalities early)  Most  least frequent: anemia, paresthesias, GI complaints, glossitis (sore tongue), difficulty walking Lab:  Macroovalocytes of RBC & hypersegmentation of granulocytes;  Hypercellular bone marrow with lots of erythroid precursors  Hemolysis: ↑LDH, hyperbilirubinemia, ↑ Fe

DDx: megaloblastosis: Interference with DNA synthesis  Chemotherapeutic drugs  Acute leukemia  Rare inherited disorders (B12 or folate deficiency too!)

Wikipedia on DCH:
Hodgkin's scientific mentor Professor John Desmond Bernal greatly influenced her life both scientifically and politically. He was a distinguished scientist of great repute in the scientific world, a member of the Communist party… She always referred to him as "Sage" and loved and admired 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 9

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 2. Peripheral / central nervous system disease
a. Classic presentation: “combined system degeneration”  dorsal/lat column probs (↓position / vibratory sense),  peripheral neuropathy,  cortical abnormalities (“megaloblastic madness”)

Schilling test: Pernicious anemia
1. Phase 1: a. Give oral radioactive cyanocobalamin + bolus dose unlabeled B12 to block tissue binding in B12-deficient individuals b. 24h urine collection (need to take up B12 to get radioactivity into urine instead of feces). c. Abnormal (<7% in urine) if IF production impaired by Ab (PA), parietal cells not working (no IF), or terminal ileum messed up (no IFreceptors) 2. Phase 2: 5 days later a. Same process but administer with oral IF; if the defective absorption is corrected, Dx = pernicious anemia (if no history of gastrectomy).

3. Methylmalonic acidemia (see above) 4. Abnormal Schilling test Causes Acquired deficiency state: 1. Decreased absorption a. Loss of intrinsic factor
(gastric atrophy, autoimmune-associated gastric atrophy is #1, also Ab against intrinsic factor / gastrectomy)

b. Terminal ileum disease
(ileal resection, gluten-induced problems, non-tropical sprue, cancer, granulomatous lesions, regional enteritis, bacterial overgrowth)

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 10

 

Impaired deconjugation or diffuse intestinal disease can lead to malabsorption Blocked utilization (cancer chemotherapy) too Diagnosis of folate defiency  ↓ serum folic acid (<3ng/mL)  ↓ RBC folate levels (<135 ng/mL)  Responds to physiologic doses of folate

Treatment: 1 mg folic acid PO qd  If caused by methotrexate, coadminister leucovorin (FH4)

Compare & contrast: B12 vs Folate
Megaloblastic anemia Combined system degeneration Dietary Deficiency Dietary Source Deficiency induces hyperhomocysteinemia Deficiency induces ↑ methylmalonic acid Site of absorption Intrinsic Factor required B12 Yes Yes Rare Muscle, liver, milk, eggs Yes Yes Terminal ileum Yes FOLATE Yes

Common Liver, leafy greens Yes

Small bowel

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 N5,10-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. 11

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 o Hard to tell in peripheral blood smear: use supravital staining (methylene blue) to clump ribosomes / mitochondria 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)

 

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


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

DIRECT COOMB’S TEST Detect antibody attached to patient’s RBC surface Use agglutinating antibody or labeled antihuman IgG

Positive for autoimmune hemolytic anemia


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


Hemoglobin chains & forms Genes
β-globin cluster α-globin cluster Chromosome 11 16 Genes Beta(β), Delta (δ), Gamma (γ), Epsilon (ϵ) Alpha (α), Zeta (ζ) HEMOGLOBIN TYPES: QUICK GUIDE Hb A (“adult hemoglobin”) Hb A2 (“minor adult”) Hb F (“fetal hemoglobin”) HbE (“embryonic hemoglobin”) Produced only just after conception α2β2 α2δ2 α2γ2 α2ϵ2 ζ2ϵ2

To make a hemoglobin: pick one from each cluster

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 o From this point on, it’s all α chains and no more ζ 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)


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 AA SS AS SC Phenotype Normal Sickle cell disease Sickle cell trait: no sickling in vivo; same survival as AA SC disease (compound heterozygote); less sx than SS

 Hemolytic anemia (shortened red cell survival)  Complex pathophysiology  variable severity  “Gene-switching” therapy

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!) 4. Classic theory: Rigid RBC  obstruction of blood flow  tissue hypoxia  tissue damage (localized pain + swelling); repeated low grade obstruction  organ damage (spleen, lungs infarct)

SC crisis:
 Sudden in onset  Unpredictable (and 80% SS have < 1 crisis/yr)  Variable: even among individuals with
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. 2. Put drop of blood in sodium hydrosulfite solution RBC lyses, releases Hb o Hb A goes into solution (clear) o Hb S precipitates (insoluble  cloudy) Sickle Prep Fast Nope Hb electrophoresis Slow Yep

3. ALL THREE TYPES (SS, AS, SC) ARE POSITIVE (just screening for presence of Hb S) – CAN’T DISTINGUISH Hb Electrophoresis 1. Run sample on electrophoresis; measure migration 2. CAN DISTINGUISH SS vs SC vs AS vs AA
Speed Can distinguish AA/AS/SS/SC

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

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 Heterozygote Homozygote Name Thalassemia minor, thalassemia trait Thalassemia major (“Cooley’s anemia”) Thalassemia intermedia Features Small RBCs, minimal anemia Transfusion-dependent (severe anemia) Microcytic / hemolytic anemia Thal major with 5+ alpha genes = more mild Thal minor with 4- alpha genes = more severe

Compound heterozygote

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


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


Background: blood under pressure; breaks in vascular continuity  exsanguination (need to seal it off)  Procoagulants: Platelets & coagulation proteins work together to stop bleeding
1. 2. 3. Vessel walls contract (reduce vascular flow  ↓bleeding) Platelets adhere (vWF, etc.), provide phospholipid-rich environment for coagulation factors Cross-linked fibrin network (via coagulation cascade)

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

General principles: basic goal is to get to formation & cross-linking of fibrin  Extrinsic pathway more important in initiation
o o o Requires “extrinsic” tissue factor Produces some thrombin (IIa) 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)


The Coagulation Cascade
Surface kallikrein HMW kininogen

Intrinsic Pathway

Extrinsic Pathway




Ca++ Phospholipid


Ca++ Phospholipid Ca++





Va fibrinogen


Ca++ Phospholipid


fibrin monomer IIa XIII XIIIa

fibrin clot

Serine proteases Cofactors XII, XI, X, IX, VII, II (prothrombin), prekallikrein  VIII (cofactor for IX)  V (cofactor for X)  HMWK* ( cofactor for XI & prekallikrein)  Tissue factor (cofactor for VII) Fibrinogen XIII Circulate in blood as zymogens, cleave & activate others Increase reaction kinetics (1000 fold) by localizing / concentrating partners to membrane surfaces (optimize reaction) Becomes insoluble (fibrin) after thrombin cleaves it  Cross-links fibrin strands covalently  Links α2-antiplasmin to fibrin | (inhibits plasmin, part of fibrinolytic system)  If no XII: clots fall apart  Need vitamin K for synthesis (warfarin inhibits)  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

Glycoprotein Transglutaminase


 II, VII, IX, X  Proteins C, S


Steps of Coagulation Cascade (in more detail)
Step 0 1 2 3 Step
0 1 1.5 2 3 3.5 4 5

Description Factor VII normally circulates; small amounts of VIIa also in blood (VII needs vitamin K) Vascular damage  Tissue factor exposed in subendothelium (normally hidden) Factor VIIa forms complex with tissue factor VIIa + TF cleaves X  Xa (X requires vitamin K)

Requirements Ca PLs Vit K

Anticoagulant Target?

Associated Disease State?

Factor XII activated by exposure to highly negatively charged surfaces (e.g. endothelium) Note: Not important in vivo (just for assays) XIIa activates prekallikrein  kallikrien; high molecular weight kininogen is a cofactor (concentrates prekallikrein on the membrane where XIIa generated) Kallikrein activates more XII  XIIa (positive feedback) XIIa also activates Factor XI (HMWK is cofactor, concentrates XI on membrane) XIa activates Factor IX  IXa ++ (needs Ca and phospholipids – surface of activated platelets; IXa needs Vit K for synth) Meanwhile, VIII activated by thrombin (Factor IIa)  VIIIa VIIIa + IXa form “tenase” complex, assembles on PL-rich platelets using Ca VIIIa+IXa (tenase) cleaves X  Xa +2 (X also requires vit K, binds to PL using Ca using γ-carboxy-glutamic acid side chains)

Requirements Ca+2 PLs Vit K

Anticoagulant Target?

Associated Disease State?

ATIII (XIa  XIi) Proteins C/S (VIIIaVIIIi)

Hemophilia B: Factor IX deficiency Hemophilia A: Factor VIII deficiency Von Willebrand Disease


0 1 2 3 4 5 4

XXa either by VIIa/tissue-factor (extrinsic) or IXa/VIIIa (intrinsic) (X needs vitamin K, happens on platelet surfaces with Ca + PLs) V  Va via thrombin (IIa) activation Va + Xa (prothrombinase complex) cleaves prothrombin (II)  thrombin (IIa) (thrombin needs Vit K for synthesis) Thrombin (IIa) cleaves fibrinogen  fibrin monomers Fibrin monomers polymerize (loose fibrin clot, hydrostatic bonds Thrombin activates XIII  XIIIa XIII is a transglutaminase; catalyzes fibrin cross-linking & affixes α2-antiplasmin

Requirements Ca+2 PLs Vit K

Anticoagulant Target?

Associated Disease State?

Prot. C/S (VaVi) ATIII (Xa  Xi) ATIII (IIa  IIi)

Factor V Leiden (hypercoaguable) Thrombin deficiency = embryonic lethal


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) YES (VII) no Activated Partial Thromboplastin Time (APTT, PTT) no YES (XII, HMWK, prekallikrein, XI, VII) YES (X, V, II, fibrinogen) NEITHER (need test of clot strength; these are just for clot formation)

Extrinsic Pathway Intrinsic Pathway Common Pathway Factor XIII

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

Hemophilia A: congenital factor VIII deficiency  X-linked, recessive (1/10k males)  Deep tissue bleeds, hemarthroses (joint bleeds)  Severity varies with factor VIII level (mild > 5%, moderate 1-5, severe >1%) Hemophilia B: congenital Factor IX deficiency  X-linked inheritance (1/50k males)  Similar manifestations to hemophilia A Treatment of hemophilia A:  Recombinant/plasma-derived factor VIII  DDAVP (desmopressin) to release VIII & vWF (mild only) Treatment of hemophilia B:  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  Immature forms of II (prothrombin), VII, IX, X o Also C and S  Half-lives vary: VII has shortest half-life  prolonged PT 1st (extrinsic) o Eventually PT & APTT both abnormal  Treatment: Vitamin K (oral, sub-q, IV)

Etiologies of Vit K deficiency:  broad-spectrum abx  surgery  poor nutrition  excessive biliary drainage  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


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

Circulation & Endothelial Cells: Anticoagulants
Circulation: prevent local accumulation of activated coagulation factors & incidental thrombous formation Endothelial cells:  express thrombomodulin (binds thrombin, activates protein C)  don’t express tissue factor  produce prostacyclin (PGI2): inhibits platelet function  promote fibrinolysis (tissue plasminogen activator: activates plasminogen)

 platelets  coagulation factors

    circulation endothelial cells fibrinolytic system endogenous anticoagulant proteins

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 Plasmin Plasminogen Plasmin: cleaves fibrin into fragments (fibrin degradation products, FDP)  Opposed by α2-antiplasmin (connected to clot surface by Factor XIIIa); prevents premature clot lysis
Plasminogen activator inhibitor I

Fibrin 2-antiplasmin



Disease states
Pathological venous thrombosis: imbalance between prothrombotic/antithrombotic mechanisms; congenital/acquired  Usually need several risk factors Acquired risk factors for venous thrombosis (risk adds up, then you go over the top & clot)  Immobility

Factor V Leiden: congenital risk factor for venous thromb
  Mutation (ARG 506 GLU) in the spot where APC cleaves V to inactivate it End result: Factor V resistant to APC (↑ clotting)

(↓ clearance of coagulation factors)

 Estrogens / OCP
(↑ synthesis of clotting factors VIII, vWF, fibrinogen; ↑ c4b binding protein  ↓ free protein S)

Epidemiology: 5% US Caucasians, up to 15% Swedish are heterozygous; uncommon in other groups.  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

   

Surgery/trauma Older age (↑ coagulation factor levels) Pregnancy (like estrogen therapy) Cancer (hypercoagulable; ↑tissue factor production?)

Lab tests:  Activated protein C resistance assay (functional test) timewithout APC o Add APC to patient plasma diluted 1:5 in factor V deficient plasma, do APTT, calculate time  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
with APC

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) 26

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)

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


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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