• What is hemostasis?
o Rapid, localized process; balance bw bleeding and clotting
o Body stops bleeding after injury, maintains blood in fluid state in vascular compartment
o Controlled activation of plts and soluble clotting factors, form stable fibrin clots
• What are the major constituents of the hemostatic pathway?
o Coagulation proteins, plts, endothelium
o Each interacts and influences all others
• What are the 3 simultaneous pathways that cause stable hemostatic plug after BV injury?
o Neural → BV constriction → reduced blood flow
o TF → coagulation activation → thrombin, fibrin
o Plt activation → primary hemostatic plug → plt fusion
• What are the 2 stages of hemostasis?
o Primary: response to BV injury, form plt plug adhere to endothelium, immediately limit bleeding
o Secondary: stable clot formation, enzymatic activation of coagulation proteins, produce fibrin, reinforce plt plug, gradual fibrinolysis dissolves plug
• What is the main purpose/advantage of the endothelium vascular system?
o Smooth endothelium helps blood flow
o Intact endothelial cells inhibit plt adherence and coagulation
o Endothelial injury promotes local clot formation
• What role does vasoconstriction play in hemostasis?
o Narrows lumen to reduce blood loss
o Brings plts and proteins closer to vessel wall
o Enhance contact activation of plts (VWF, collagen, plt membrane glycoprotein Ib)
o Activated plts enhance activation of coagulation proteins
• How quickly does coagulation occur after BV injury?
o Normally, coagulation initiated within 20 seconds of injury
• What are plts?
o Small, anucleated, cytoplasmic fragments of megakaryocytes
o Normal: 150-400 x 10E9 /L
o Live 9-12 days
o old cells removed by spleen and liver
o ~2/3 circulate in peripheral blood
o ~1/3 sequestered in spleen (2 pools constantly exchanging, equilibrium)
o Spontaneous hemorrhage <10 x 10E9 /L
• Where do plts come from?
o released from BM by megakaryocytes
o Megakaryocyte proliferation stimulated by thrombopoietin (TPO)
o TPO: humoral factor; produced by liver, kidney, spleen, BM; made at constant rate
• How does plt adhesion happen?
o Damaged endothelium exposes blood to subendothelial (SE) tissue matrix w adhesive molecules
o Plt receptor GPIb binds SE collagen fibers via VWF
o Plt adherence stops initial bleeding
• What is VWF?
o Multimeric glycoprotein
o Parts made in endothelial cells and megakaryocytes
o Stored in endothelial cells (Weibel-Palade Bodies) and Plt alpha-granules
o Circulate in plasma, 7-10 mg/mL
o Ligand for Plt receptor Ib/IX
• What happens to plts after aggregation?
o Undergo a shape change
o Oval → dendrites → more dendrites → fried egg
• What things are necessary for plt-plt interaction in aggregation?
o Dense granule release from adhering plts
o Requires Ca2+ and ATP; fibrinogen; fibrinogen recptors GPIIB and IIIA
• What plt-plt interaction happens in aggregation?
o Begins 10-20 sec after injury and adhesion
o ADP released from plt cytoplasm → expose GIIb and GIII, fibrinogen binds
o EC Ca2+-dependent fibrinogen bridges form bw plts, promote agg
o = primary/reversible aggregation
o 2nd/irreversible: begins w release of dense granules
• What is the purpose of secretion of dense granules?
o Begin 2nd agg
o Have lots of ADP, binds plt membrane triggering synthesis and release of TXA2
o Amplifies initial plt agg into big plt mass
o = primary hemostatic plug
• What is the primary plt plug?
o Dt plt interaction with vessel wall, each other
o Plug: fragile, can easily be dislodged from vessel wall
• What are some inherited plt defects?
o Bernard soulier dz: def GPIb o Von Willebrand dz: def VWF o Glanzmann’s Thrombasthenia: GPIIb, GPIIIA o Storage pool dz: dense body o Gray plt syndrome: alpha granule o Defective procoagulant activity o COX deficiency: dense tubular system
• What is von Willebrand dz?
o Most frequent inherited bleeding disorder, ~1/100
o many clinical manifestations
o Clinically significant vWD : 125/1 million
o Severe dz in 0.5-5/ 1 million
o Autosomal inheritance pattern
• Who discovered vWD? What did he find? Future findings?
o 1931-Erik von Willebrand o Hereditary pseudohemophilia o Prolonged BT, normal plt ct o Mucosal bleeding o M=F o 1950s: prolonged BT assoc w def fx VIII o 1970s: discover VWF o 1980s: VWF gene cloned
• What is the classification of vWD?
o Type I: partial quant def; 70%; mild-mod dz
o Type II: qual def; 25%; mild-mod dz
o Type III: near/total def; 5%; severe dz
o Additional subclass: acquired vWD
• What is bleeding time? Recommended criteria prior to testing?
o Test adequacy of primary hemostasis or plt fxn
o 1. Plt ct: >75,000/mm3
o 2. Only I BT/24 hrs, unless excuse
o 3. stop heparin 6 hrs prior
• How is bleeding time done?
o BP cuff on arm (@40 mm Hg)
o 2 incisions made on inner forearm
o Blot w filter paper every 30 sec
o Record time for clotting to occur (bleeding stops)
• What are sources of error giving false + results in BT?
o BP cuff too high (>40) or too low o lo fibrinogen (<100 mg/dl) or plt ct (100,000 /mm3). o Drugs that affect plt fxn (aspirin) o Incision too shallow/deep o disturb clot w filter paper
• what are the ref ranges for BT?
o 1-3 mins (duke method)
o 2-7 mins (template Ivy method)
• What is clinical sig of BT?
o Usu very long in congenital or acquired plt defects
o Thrombasthenia, Bernard-soulier, storage pool dz, aspirin, VWD, uremia, liver failure, plt ct <75,000
• What is the plt fxn screen?
o Replaces BT as a test of plt fxn
o PFA-100; ordered as “plt fxn screen”
o Blue top tube
o Measures time for blood to block membrane coated w collagen/epi or col/ADP
• How would you interpret plt fxn screen results?
o Norm epi, N ADP: normal plt fxn
o Abn epi, N ADP: “aspirin effect”
o A epi, A ADP: abn plt fxn, valvular heart dz, renal failure, VWD
• What is aspirin’s role in primary hemostasis?
o =acetylsalicylic acid
o Limits blood clotting
o = “blood thinner” for ppl at risk for life-threatening clots
o Inhibits plt activation by irreversible inhibition (acetylation) of plt enzyme COX, inhibiting TXA (need for plt agg, adhesion unaffected)
o Lasts at least 7 d (lifetime of plt)
o So pts should avoid aspirin 7 d if doing plt fxn test
o Also acetylate fibrinogen, alter clot formation, enhance fibrinolysis
• What is 2nd hemostasis?
o Coagulation factors form fibrin strands, strengthen plt plug
o Activated plt release granules, stim more activation, enhance hemostasis
• What is the coagulation cascade?
o Contact activation pathway (intrinsic)
o Tissue factor pathway (extrinsic), primary for initiation of coagulation
o Zymogen of serine protease and its glycoprotein co-factor activated…
• What are clotting factors, and how are they named?
o Circulate in zymogen/inactive forms
o Roman numeral (order of discovery)
o “a” once activated
• What are the numbers, names, source, and pathway of all the clotting factors?
o I- fibrinogen, liver, common
o II- prothrombin, liver (vit K dep), common
o III- tissue factor/thromboplastin, released by damaged cells/extrinsic, by plts/intrinsic
o IV- calcium ions, bone and gut, entire process
o V- proaccelerin/labile factor, liver and plts, E and I
o VI- dne
o VII- proconvertin/stable factor, liver (K), E
o VIII- anti-hemolytic factor, plts and endothelium, I
o IX- Christmas factor, liver (K), I
o X- stuart-prower factor, liver (K), E and I
o XI- plasma thromboplastin antecedent, liver, I
o XII- Hageman factor, liver, I, also activates plasmin
o XIII- fibrin-stabilizing factor/VWF, liver, slows fibrinolysis
• What is the extrinsic/TF pathway?
o Initiated by release of tissue thromboplastin (TP, III), exposed to blood after vessel injury
o Circulating VII forms complex w TP and Ca → VIIa
o Rapidly converts X → Xa
o Xa and Va catalyze prothrombin (II) to thrombin (IIa), to convert fibrinogen (I) to fibrin
• What is the intrinsic pathway?
o Initiated after exposure to neg charge foreign substances (collagen, subendothelium, phospholipids)
o XII → XIIa
o W contact factors (prekallikrein, kininogen), XI -→XIa
o IX → IXa, in presence of Ca2+
o IXa, VIII, protein C, Ca2+, PF3 (source of phospholipids) =tenase complex
o → Xa, → thrombin
• What is the common pathway?
o Fibrin clot formation
o End products of intrinsic or extrinsic, X → Xa
o Xa and Va: II → IIa (thrombin)
o Thrombin enzymatically converts fibrinogen → fibrin monomer, polymerize into insoluble strands
o Thrombin activates XIII → XIIIa → crosslinks, stabilize fibrin clot
o This occurs on plt surface
• What is thrombin? Its roles?
o Central role of bioregulation of hemostasis in normal and pathologic conditions
o Potent plt agg and secretion agent
o Amp coagulation by activating V, VIII, XI (Activating V and VIII = positive feedback to amplify more thrombin)
o Make XIIIa for fibrin stabilization (soft vs hard clot)
o Complex w endothelial thrombomodulin to activate protein C (Active C inactivates Va, VIIIa, slow thrombin formation)
o Stimulation of tissue repair
• What are the 3 phases of the coagulation cascade?
o Initiation: injury, blood contacts SE, TF exposed, VII → VIIa, activate IX and X, Xa binds Va on cell surface
o Amplification: Xa/Va convert little pro → thrombin, activates VIII, V, XI and plts locally, XIa activates IXa, activated plts bind Va, VIIIa, IXa
o Propagation: VIIIa/IXa activates X on plt surface, Xa/Va convert lots of pro → thrombin (“thrombin burst”) → stable fibrin clot
• What are the d/os of 2nd hemostasis? Sxs? sxs common to 1st and 2nd hemostatic dos?
o Dos of proteins of fibrin formation, or assoc w fibrinolysis
o Sxs: delayed bleeding, deep mm bleed, spontaneous jt bleed
o Common: Ecchymoses, GI bleed, hematuria, hypermenorrhea, gingival bleed, inc bleed after tooth extraction, intracranial bleed, epistaxis
o Mb deep spreading hematomas, bleed into jts, hematuria, dt failure to reinforce plt plug
• What is prothrombin time (PT)? results?
o Evaluates extrinsic path
o Prolonged: def VII, X, V, II (prothrombin), or I (fibrinogen)
o Or: vit K def (needed for synthesis of II, VII, IX, X)
o Liver dz, warfarin, DIC, excessive heparin
• What is the international normalized ratio (INR)? Effects of heparin?
o PT value = patient PT/ PT for control plasma
o INR= correction factor applied (international sensitivity index)
o = (PT pt / PT control) ^ISI
o 1.0= pt’s PT is normal
o > 1.0 = clotting time elevated
• What is the therapeutic interval for oral anticoagulant therapy? Application?
o 2.0-4.5 (target 2.0 – 3.0)
o 3 = hemorrhagic
o App: monitor oral anticoag tx (warfarin, etc)
o Heparin won’t prolong INR
o For heparin tx, monitor aPTT or aPTT ratio
• What is coumadin (warfarin, dicoumarol)?
o Oral anticoagulant
o Takes few days for effects to show
o Inhibit production of vit k dep factors (II, VII, IX, X) and Protein C and S
o Monitored w PT (since VII has shortest 1/2 life and becomes deficient first)
• What is activated partial thromboplastin time (aPTT)? Results?
o Eval intrinsic
o Prolonged aPTT, normal PT: def VIII, IX, XI, or XII
o Prolong both aPTT and PT: def X, V, II, or I (all rare)
o Normal aPTT: XIII def or VII def (PT prolonged)
o Heparin: most common cause of prolonged aPTT
• What is heparin?
o Administered IV
o Immediate inhibition of blood clotting
o Accelerates action of ATIII to inactivate IIa (thrombin)
o Monitored w PTT
• What are normal PT and aPTT times?
o PT: 9.9-13 sec
o aPTT: 25-35 sec
• what are the types of dos of proteins of fibrin formation? Tests?
o Hereditary vs acquired
o Quantitative vs qualitative deficiencies (not differentiated by PT or aPTT)
o Qualitative: prolong clotting test, found by Ig tests
o Activity assays are essential to screen for deficiencies
• What is thrombin time?
o Measures conversion of fibrinogen to fibrin
o Add thrombin to pt’s plasma
o Measure time to clot
• What are some factor assays used for?
o IX: x-linked Hemophilia B
o VIII: X-linked Hemophilia A
• What are the hemophilias?
o A: VIII def, antihemophilic factor, x-link recessive, most common
o B: IX, Christmas factor (family name), x-linked
o C: XI, AR, Ashkenazi, mild-severe sxs
• How is hemophilia diagnosed?
o Readily: in severe dz, or FHx
o Dx: unusual bleeding sxs early in life (age varies w severity), FHx, PE, Lab eval
• Congenital def: Plt N, PT N, aPTT N, PFA N, TT N:
o XIII, mild def of any factor, plasminogen activator inhibitor-1, a2 anti-plasmin
• Congenital def: Plt N, PT A, aPTT N, PFA N, TT N:
• Congenital def: Plt N, PT N, aPTT A, PFA N, TT N:
o XII, XI, IX, VIII, prekallikrein, high molecular weight kininogen
• Congenital def: Plt N, PT A, aPTT A, PFA N, TT N:
o X, V, II
• Congenital def: Plt N, PT A, aPTT A, PFA N, TT A:
• Congenital def: Plt N, PT N, aPTT N/A, PFA N/A, TT N:
• What are acquired coagulation dos?
o More common that hereditary o Usu defects of mult factors o Often bleeding from mult sites o Mb dt another dz o Many mechanisms
• What is DIC?
o Altered hemostasis
o Uncontrolled formation and lysis of fibrin in vessels
o Systemic, rather than local site of injury
o Fibrin deposited diffusely in capillaries, arterioles, venules
o Factors/plts/proteins consumed faster than synthesized
o Too much clotting followed by too much bleeding
• What is vit k? def?
o Lipophilic, hydrophobic; posttranslational modification of II, VII, IX, X, C, S
o Quinone: green leafy veg, fish, liver, intestinal organisms (B fragilis, E coli)
o Adds 2nd C=O group to y carbon of glutamic acid at end of II, VII, IX, X (pocket for Ca2+ promotes phospholipid binding)
o Def: mb dt warfarin (antagonist); non-active II, VII, IX, X, C, S
• What may cause K def? tx?
o Malnutrition, biliary obstruction, malabsorption, Abx tx
o Tx: vit k, fresh frozen plasma
• Why is excessive clotting inhibited? How?
o Would be dangerous
o Clotting factors rapidly inactivated by circulating proteins
o So clotting only at site, not systemically
o Fibrin inhibits thrombin = neg feedback loop
• How is fibrinolysis activated?
o Gradual enzymatic cleavage of fibrin to soluble fragments
o Limits extent of hemostatic process, re-establish normal blood flow
o Important: local activation of plasminogen (PLG) to plasmin
o Plasmin enzyme system =fibrinolytic system
• What is plasmin?
o Responsible for degradation of fibrin (or fibrinogen)
o → fibrin degradation products (FDPs)
o Sites of plasmin cleavage: similar in fibrin and fibrinogen
o Also destroy V, VIII, others
• What is the Proteins C and S regulatory pathway of coagulation?
o Excess thrombin binds thrombomodulin on endothelium surface → activate C
o C + S → complex degrades Va and VIIIa -> clotting stays local
o C and S are vit K dep
• What is the antithrombin III regulation pathway?
o Inhibit thrombin (IIa) o Inactivate Xa o In most cells, esp basophilic granules and plt surface o Limited anticoag effect on its own o Activity ↑ 1000x w heparin
• What is factor V Leiden mutation?
o 3-8% of Caucasians
o Resistant to degradation by C-S complex
o → hypercoagulable state, risk venous thrombosis
o Women w OB complications: preeclampsia, still-born, IU growth retardation, repeat preg losses
o Females: BCP inc risk of thrombosis 35x
• What happens w imbalance of hemostasis?
o Thrombi: excessive local/systemic coag activation
o Sustained bleeding: excess local/systemic fibrinolytic activity
• What is usu cause of delayed hemostasis? Sx?
o Either plt do or coag defect
o Imbalance bw: abn slow hemostasis, normal rate fibrinolysis
o Bleeding episode mb prolonged
• What happens with an inadequate fibrinolytic response?
o May retard lysis of thrombus, or contribute to extension
• What are the 3 categories of clotting/bleeding dos?
o Vascular and plt dos
o Coag factor def, or specific inhibitors
o Fibrinolytic dos
• What tests are used to differentiate clot/bleed dos?
o Plt ct o Peripheral blood smear o Ivy BT (N=2.5-9.5 min) or PFA o PT- contains thromboplastin and CaCl, extrinsic and common (N=11-13 sec) o aPTT: contact activators, plt sub, CaCl added, intrinsic and common (23-35 sec, varies) o TT: add thrombin, common (15-22 sec) o PT and aPTT abn studies o Coag factor assays
• What would you do after PT and aPTT studies were abn?
o mix pt plasma and normal plasma, distinguish bw factor def and coag inhibs
o if assay is corrected: dt fx def
o if partially corrected or uncorrected: dt inhibitor
• what are CBC and smear tests for bleeding dos?
o Plt ct: thrombocytopenia
o RBC and plt morph: TTP, DIC, etc
• What are coagulation tests for bleeding dos?
o PT: extrinsic/common pathways
o PTT: intrinsic/common pathways
o Coagulation factor assays: specific factor deficiencies
o 50:50 mix: inhibitors (like Abs)
o Fibrinogen assay: dec fibrinogen
o TT: qual/quantitative fibrinogen defects
o FDPs or D-dimer: fibrinolysis (DIC)
• What are the plt fxn tests for bleeding dos?
o VWF: VWD
o BT: in vivo test (non-specific)
o PFA: qualitative plt dos and VWD
o Plt fxn tests: qualitative plt dos
• Swollen tender knee, after falling. Hx prolonged bleeding after tooth extraction. Never hospitalized. No meds. PT=10sec (8-14). aPTT=57sec(24-36). Plt=450k (130-350k). mix PTT=32 sec (24-36). VIII=5% (50-150). IX=132%. XI=112%
o Hemophilia A= factor VIII deficiency