PRIMARY HEMOSTASIS

1. Vasoconstriction (immediate)
   • Neurogenic reflex + local endothelin release (potent vasoconstrictor from endothelium)
   • Reduces blood flow to injured area - transient; bleeding resumes without platelet activation
2. Subendothelial Exposure
   • Vascular injury disrupts endothelium and exposes subendothelial collagen
   • von Willebrand factor (vWF) - present in subendothelial matrix AND plasma - binds to exposed collagen
3. Platelet Adhesion
   • vWF acts as a bridge between subendothelial collagen and platelet surface receptor GpIb
   • Deficiency of vWF = von Willebrand disease; deficiency of GpIb = Bernard-Soulier syndrome
4. Platelet Activation (Shape Change + Granule Release)
   • Platelets change from smooth discs to spiky "sea urchins" - greatly increased surface area
   • Shape change triggers conformational change in GpIIb-IIIa increasing its affinity for fibrinogen
   • Negatively charged phosphatidylserine translocates to the platelet surface - serves as nucleation site for coagulation factor assembly
   • Alpha (α) granules release: fibrinogen, factor V, vWF, fibronectin, PDGF, platelet factor 4, TGF-β
   • Dense (δ) granules release: ADP, ATP, calcium, serotonin, epinephrine
   • Thrombin (from secondary hemostasis) activates platelets via protease-activated receptors (PARs)
5. Platelet Aggregation
   • Released ADP + produced thromboxane A2 (TXA2) recruit additional platelets
   • GpIIb-IIIa on adjacent platelets binds fibrinogen bridges - platelet-to-platelet aggregation
   • Deficiency of GpIIb-IIIa = Glanzmann thrombasthenia
   • Result: Primary hemostatic plug is formed

SECONDARY HEMOSTASIS

1. Tissue Factor (TF) Exposure
   • TF is a membrane-bound procoagulant glycoprotein on subendothelial cells (smooth muscle cells, fibroblasts) - not normally exposed to blood
   • Vascular injury exposes TF to circulating factor VII → forms TF/VIIa complex
2. Extrinsic Pathway (initiates coagulation in vivo)
   • TF + Factor VIIa complex activates Factor X and Factor IX
   • Assessed by PT (Prothrombin Time) - tests factors VII, X, V, II, fibrinogen
   • Prolonged PT: factor VII deficiency, warfarin therapy, liver disease
3. Intrinsic Pathway (amplifies coagulation)
   • Factor IXa + Factor VIIIa complex (on platelet phospholipid surface) = most important activator of Factor X in vivo
   • Assessed by aPTT (Activated Partial Thromboplastin Time) - tests factors XII, XI, X, IX, VIII, V, II, fibrinogen
   • Prolonged aPTT: hemophilia A (VIII deficiency), hemophilia B (IX deficiency), heparin therapy
4. Common Pathway
   • Factor Xa + Factor Va → converts prothrombin (II) to thrombin (IIa)
   • Thrombin is the central effector:
     • Cleaves fibrinogen → insoluble fibrin monomers
     • Activates Factor XIII → crosslinks fibrin polymers (covalent crosslinking)
     • Activates factors V, VIII, XI (amplification feedback loops)
     • Potent platelet activator via PARs
     • On normal endothelium: switches to anticoagulant function
5. Clot Stabilization
   • Factor XIII crosslinks fibrin covalently
   • Platelet contraction compacts the clot
   • Result: Solid, permanent secondary hemostatic plug

• Dilution - flowing blood washes out activated factors; liver clears them
• Platelet phospholipid requirement - limits assembly away from injury site
• Endothelial anticoagulants: thrombomodulin (binds thrombin → activates protein C → degrades V and VIII), TFPI (tissue factor pathway inhibitor), heparan sulfate (activates antithrombin III)
• Fibrinolysis: plasminogen → plasmin (via tPA from endothelium) → cleaves fibrin → generates D-dimers (clinical marker of thrombosis); plasmin controlled by α2-antiplasmin

VON WILLEBRAND FACTOR (vWF) DISEASES

• Multimeric plasma glycoprotein
• Synthesized by endothelial cells (stored in Weibel-Palade bodies) and megakaryocytes (stored in α-granules)
• In plasma: circulates as a complex with Factor VIII (protects it from degradation)
• In subendothelial matrix: acts as adhesion bridge between collagen and platelet GpIb
• High-molecular-weight (HMW) multimers are the most biologically active form

1. Platelet adhesion - GpIb receptor bridge to collagen (primary hemostasis)
2. Factor VIII carrier - shields factor VIII from proteolysis in plasma

Von Willebrand Disease (VWD)

• Most common inherited bleeding disorder (~1% of population)
• Autosomal dominant (most types)
• Clinical features: mucosal bleeding, excessive wound bleeding, menorrhagia
• Compound defect: platelet function + coagulation (but platelet defect dominates clinically in most)

Types:

• Autosomal dominant
• Quantitative deficiency - reduced amount of structurally normal vWF
• Mild decrease in Factor VIII (clinically insignificant)
• Mild-to-moderate bleeding symptoms
• Responds to DDAVP (releases stored vWF from endothelium)

• Selective loss of high-molecular-weight multimers (most active form) → functional deficiency despite normal or near-normal total vWF quantity
  • Type IIA: HMW multimers not synthesized (true deficiency of large multimers)
  • Type IIB: "Hyperfunctional" HMW multimers produced but are rapidly cleared from circulation; these abnormal multimers cause spontaneous platelet aggregation → mild chronic thrombocytopenia (platelet consumption); similar mechanism to TTP
  • Type IIM: Decreased platelet binding (without loss of multimers)
  • Type IIN: Decreased factor VIII binding → phenotype resembles hemophilia A

• Autosomal recessive (homozygous mutation)
• Virtual complete absence of vWF
• Severe bleeding; markedly reduced Factor VIII (because vWF no longer protects it) → features of both VWD + hemophilia A
• DDAVP ineffective

• Prolonged bleeding time / PFA-100 closure time
• Prolonged aPTT (in types with low Factor VIII)
• Reduced vWF antigen (types I, III)
• Reduced ristocetin cofactor activity (vWF functional assay)
• Ristocetin-induced platelet aggregation (RIPA): absent/reduced in type I; increased in type IIB (at low doses)

HEMOLYTIC ANEMIA

• Reticulocytosis (compensatory marrow response)
• Marrow erythroid hyperplasia
• Extramedullary hematopoiesis (liver, spleen, lymph nodes) in severe cases
• Decreased serum haptoglobin (binds free Hb; removed from circulation; falls in both intra- and extravascular hemolysis)

Classification Framework

A. By Site of Destruction

• Destruction by splenic and hepatic macrophages
• Cause: decreased red cell deformability → cells trapped in splenic cords → phagocytosed
• Findings:
  • Hyperbilirubinemia + jaundice (unconjugated bilirubin from Hb degradation in macrophages)
  • Splenomegaly ("work hyperplasia" of phagocytes)
  • Pigment gallstones (bilirubin-rich) if chronic
  • NO hemoglobinuria
  • Haptoglobin low (macrophages regurgitate some free Hb)

• Red cells burst within blood vessels
• Causes: mechanical forces (defective heart valve, microangiopathic changes), complement fixation (PNH, transfusion reaction), toxins (Clostridium, snake venom), heat
• Findings:
  • Hemoglobinemia (pink/red plasma)
  • Hemoglobinuria (red/dark urine)
  • Hemosiderinuria (sloughed renal tubular cells containing hemosiderin)
  • Iron deficiency if chronic (iron lost in urine)
  • Haptoglobin markedly low

B. By Defect: Intrinsic vs. Extrinsic

Key Hemolytic Anemias (Increased Destruction)

Hereditary Spherocytosis

• Mutation in membrane skeleton proteins (spectrin, ankyrin, band 3, band 4.2) → weakened skeleton-lipid bilayer interaction → red cells shed membrane vesicles → become spherical
• Spherocytes: decreased surface area-to-volume ratio, non-deformable → trapped in splenic cords → extravascular hemolysis
• Autosomal dominant (mostly)
• Smear: dark red cells lacking central pallor
• Splenomegaly prominent (500-1000 g; normal 150-200 g)
• Cholelithiasis in 40-50%
• Treatment: splenectomy corrects anemia (spherocytes persist but are no longer destroyed)

G6PD Deficiency

• X-linked recessive
• G6PD protects RBCs from oxidative damage via glutathione pathway
• Deficiency → oxidant stress (infections, drugs - primaquine, dapsone; fava beans) → Hb oxidized → Heinz bodies form → membrane damage → intravascular + extravascular hemolysis
• Episodic hemolysis, not chronic
• Smear: "bite cells" (Heinz body removed by spleen), blister cells

Sickle Cell Disease (HbSS)

• Point mutation in β-globin gene (glutamate → valine at position 6)
• Deoxy-HbS polymerizes → sickling → rigid, inflexible cells
• Vascular occlusion (painful crises, organ infarcts) + hemolysis (extravascular + intravascular)
• Smear: sickle cells, target cells, Howell-Jolly bodies (functional asplenia)

Autoimmune Hemolytic Anemia (AIHA)

• Antibody-mediated extravascular hemolysis
• Warm AIHA: IgG antibodies (peak at 37°C), DAT positive, spherocytes on smear
• Cold AIHA: IgM antibodies (peak at cold temperatures, activate complement), agglutination in periphery
• DAT (direct antiglobulin test / Coombs) positive

Microangiopathic Hemolytic Anemia (MAHA)

• Mechanical fragmentation of RBCs passing through damaged/narrowed small vessels
• Seen in: TTP, HUS, DIC, malignant hypertension, HELLP syndrome
• Smear: schistocytes (fragmented RBCs, helmet cells) - hallmark
• Intravascular hemolysis

Paroxysmal Nocturnal Hemoglobinuria (PNH)

• Acquired clonal mutation in PIG-A gene → deficiency of GPI-anchored proteins (CD55, CD59)
• CD55 and CD59 normally protect RBCs from complement
• Without them: complement activation → intravascular hemolysis (especially nocturnal, when pH drops)
• Classic triad: hemolytic anemia, thrombosis (especially portal/hepatic vein), cytopenias
• Morning hemoglobinuria is classic but not always present
• Flow cytometry (loss of CD55/CD59 on RBCs and granulocytes) is diagnostic

Malaria

• Plasmodium falciparum infects and lyses RBCs (intravascular hemolysis)
• Blackwater fever: massive hemolysis → hemoglobinuria

CLASSIFICATION OF ANEMIA

I. By MCV (Morphologic / Wintrobe Classification)

• Low MCHC (hypochromic) = iron deficiency, thalassemia, sideroblastic anemia
• High MCHC = hereditary spherocytosis (spherocytes have no central pallor = increased MCHC)
• Normal MCHC = most other anemias

• High reticulocyte count = bone marrow responding normally → blood loss or hemolysis
• Low reticulocyte count = bone marrow failure or underproduction

II. By Mechanism (Pathologic Classification)

A. DECREASED PRODUCTION (Hypoproliferative) - Low Reticulocyte Count

• Iron deficiency - most common worldwide; affects Hb synthesis → microcytic hypochromic anemia
• B12 deficiency - affects DNA synthesis; megaloblastic changes; neurologic features (subacute combined degeneration)
• Folate deficiency - affects DNA synthesis; megaloblastic (NO neurologic features)

• Aplastic anemia - pancytopenia from destruction/suppression of pluripotent stem cells (often autoimmune T-cell mediated, or from chemicals/radiation/drugs); normocytic; hypocellular marrow
• Pure red cell aplasia - selective absence of erythroid precursors

• Marrow replaced by tumor (metastatic carcinoma), granulomas, fibrosis
• Normocytic; leukoerythroblastic smear (nucleated RBCs, immature WBCs)

• Chronic kidney disease - most common cause of normocytic anemia in CKD
• Endocrine failure: hypothyroidism, adrenal insufficiency, androgen deficiency

• Chronic infection, inflammation, malignancy
• Hepcidin elevated (acute phase reactant) → sequesters iron in macrophages → iron unavailable for erythropoiesis
• Usually normocytic; can be microcytic in severe/prolonged cases
• Low serum iron, low TIBC, HIGH ferritin (contrast with iron deficiency: low ferritin, high TIBC)

• Defective heme synthesis → iron accumulates in mitochondria around erythroblast nucleus
• Pathognomonic: ring sideroblasts on Prussian blue stain of bone marrow
• Causes: inherited (X-linked, ALAS2 mutation), acquired (alcohol, lead, drugs - isoniazid), myelodysplastic syndrome

• α-thalassemia: deletion of α-globin genes (chromosome 16)
• β-thalassemia: point mutations in β-globin gene (chromosome 11)
• Ineffective erythropoiesis + hemolysis (extravascular) → both decreased production AND increased destruction

B. INCREASED DESTRUCTION (Hemolytic) - High Reticulocyte Count

• Red cell membrane: Hereditary spherocytosis, hereditary elliptocytosis
• Enzyme defects:
  • Hexose monophosphate shunt (oxidant protection): G6PD deficiency, glutathione synthetase deficiency
  • Glycolytic enzymes (energy): Pyruvate kinase deficiency, hexokinase deficiency
• Hemoglobin defects:
  • Deficient synthesis: Thalassemia syndromes
  • Structurally abnormal: Sickle cell disease, unstable hemoglobins
• Acquired intracorpuscular: PNH (GPI-anchor defect)

• Antibody-mediated: AIHA, hemolytic disease of newborn (Rh), transfusion reactions, drug-induced
• Mechanical trauma:
  • Microangiopathic: HUS, TTP, DIC, malignant hypertension
  • Cardiac: defective prosthetic valves
  • Repetitive physical trauma: march hemoglobinuria
• Infections: Malaria, babesiosis (RBC parasitized and lysed)
• Chemical/toxic: Clostridium toxins, oxidants, snake venom

Acute vs. Chronic Anemia