theme 3

hemostasis (what - three phases - ends with what?)

• process that stops bleeding after vessel injury

• three phases

  • vasoconstriction

  • primary hemostasis → platelets

  • secondary hemostasis → coagulation

• ends with fibrinolysis → clot breakdown after repair

prim hemostasis (PM) → globally what, what it produces, triggered by what

• Platelet adhesion, activation, and aggregation.

• Forms the primary platelet plug.

• Triggered by collagen + vWF exposure after endothelial injury.

steps PM (5)

1. vasocon → immediate → limit blood flow

2. platelet adhesion → GPIb-vWF (high shear), GPVI + GPIa to collagen 

3. platelet activation → shape change, granule release

4. platelet aggregation → GPIIb/IIa binds to fibrinogen + vWF 

5. plug formation 

platelet adhesion → key receptors (3+ binding to?)

• GPIb + vWF → initial tethering (high shear)

• GPVI + collagen → platelet activation

• GPIa/IIa + collagen → form adhesion 

platelet activation - what, release from what + what, production of what

• shape change → spiky/activated form

• release of granules

  • alpha → vWF, fibrinogen, FV

  • dense → ADP, serotonin

• production of TxA2 via COX → increases vasocon + aggregation 

platelet aggregation → binding of, mediated by

• binding of platelets TO platelets

• mediated by GPIIb/IIIa binding to fibrinogen and vWF

• final step forming the platelet plug

interaction of prim and secon hemostasis

• prim plug is weak and unstable

• thrombin from secondary hemostasis (SH)

  • enhances platelet acti

  • stabilizes plug by converting fibrinogen → fibrin

role endothelium in hemostatic balance when intact 

• anti-thrombotic

• NO, prostacyclin → inhibit plat aggregation

• thrombomodulin, TFPI, heparin-like molecules → anti-coagulant 

• tPA → promotes fibrinolysis 

role endothelium in hemostatic balance when damaged 

• pro-thrombotic

• exposes TF → coag activation

vWF - prod by, what it does, how does it exist

• produced by endothelial cells (WPB) + megakaryocutes (alpha granules)

• binds collagen + platelet GPIb → adhesion

• stabilizes circulating FVIII

• exists as high mol weight multimers  (HMWM) → most active 

vWF synthesis

• pre-pro-vWF produced in RER

• multimerization in Golgi

• stored in

  • weibel-palade bodies (endothelium) 

  • alpha granules (platelets)

• circulatin pool = mainly inactive LMWH’s 

vWF in an intact vessel

• plasma vWF bound to FVIII → inactive

• platelets remain inactive 

• subendothelial vWF seperate from blood

vWF in an injured vessel

• collagen + vWF become intravasculary exposed

• Active vWF binds platelets.

• Platelet activation + aggregation initiated.

VWD 

• most common inherited bleeding disorder 

• due to quantitative or qualitative vWF defects 

• leads to mucocutaneous bleeding

VWD - types

• type 1 → partial quantitative deficiency 

• type 2 → qual defects 

  • 2A → low HMWM + platelet binding

  • 2B → high affini for GPIb, loss of HMWM; thrombocytopenia

  • 2M → low platelet/GPIb binding, multimers normal 

  • 2N → low binding to FVIII → low FVIII

• type 3 → near total absence of vWF + very low FVIII

VWD - first level diagnostics

• vWF:Ag

• vWF:Act

• FVIII:C

VWD symptoms

• easy brusing, mucosal bleeding, epistaxis

• menorrhagia

• post-op bleeding

• if type 3 → joint/muscle bleeds bc FVIII very low

second level diagnostics

• RIPA

• vWF multimer analysis

• vWF-FVIII binding assay 

• vWF collagen binding assay 

VWD treatment 

• general → tranexamic acid, hormonal therapy (HMB)

• DDAVP

  • release endogenous vWF + FVIII

  • works best for type 1 → do not use for type 2B

  • risk of hyponatremia

• vWF concentrates (+ FVIII) → for type 3, surgery + non responders

platelets

• pragments of megakaryocytes.

• 150–450 ×10⁹/L (normal).

• Lifespan 7–10 days.

• Platelet count < 30–50 increases bleeding risk.

Thrombocytopenia → count, Causes = 3

• Platelet count <150 ×10⁹/L

• causes

  • reduced production → leukemia, aplastic anemia, chemo

  • increased destruction → ITP, DIC.

  • sequestration → splenomegaly 

  • Loss/consumption → massive bleeding.

• bleeding if <50, severe if <30

platelet function defects → causes = 3

• Congenital

  • receptor defects

  • granule defects.

• Acquired

  • uremia,

  • liver disease.

• Drug-induced: aspirin, NSAIDs, P2Y12 inhibitors.

Bernard–Soulier Syndrome (BSS)

• GPIb-IX-V defect → adhesion defect.

• Macrothrombocytopenia + mucocutaneous bleeding.

• Diagnosis: PFA, platelet aggregation abnormal with ristocetin.

• Treatment: platelet transfusion, tranexamic acid, rFVIIa.

Glanzmann Thrombasthenia

• GPIIb/IIIa defect → aggregation defect.

• Platelet count normal.

• Severe mucosal bleeding.

• Treatment: platelet transfusion, rFVIIa.

storage pool disease 

• Reduced α- or dense-granule content.

• Impaired secretion → poor aggregation + activation.

• Variable bleeding tendency.

Acquired platelet dysfunction

• renal failure (uremia)

• liver cirrhosis

• drugs

  • COX inhib → low TxA2

  • P2Y12 inhib (clopidogrel, prasugrel, ticagrelor)

  • GPIIb/IIIa inhibitors (tirofiban, eptifibatide).

Platelet Function Analyzer (PFA-100)

• screening test for platelet dysfunction + VWD.

• Measures closure time under high shear.

• Abnormal → follow with aggregation studies (LTA)

secundary hemostasis = SH

• coag cascade → generates thrombin

• thrombin converts fibrinogen → fibrin 

• fibrin stabilizes primary platelet plug 

• requires phospholipid surface (platelets) + Ca 

coag cascade branches (pathways)

• intrinsic

• extrinsic

• common

intrinsic pathway factors

• XII → XI → IX → VIII → X

extrinsic pw factors

• VII → X (via TF)

common pw factors

• X → V → II (thrombin) → I (fibrin)

intrinsic pathway → how activated, measured

• activated by nega charged surfaces (like collagen)

• measured by aPTT

  • prolonged in HA/HB + int factor deficiencies

  • heparin therapy affects aPTT

extrinsic pathway → how activated, measured

• triggered by TF exposure from damaged endothelium

• measured by PT/INR → prolonged in

  • vit K def

  • warfarin therapy

  • liver failure  

common pathway → more about factors + measurements

• Factor X → Xa

• Xa + Va → converts prothrombin → thrombin

• Thrombin converts fibrinogen → fibrin

• Prolonged PT and aPTT if defective.

thrombin functions (4)

• converts fibrinogen → fibrin.

• Activates platelets.

• Activates factors V, VIII, XI → amplifies cascade.

• Activates factor XIII → cross-links fibrin (stable clot).

factor XIII → stabilizes what, by doing…

• fibrin-stabilizing factor 

• crosslinks fibrin polymers → stable clot 

• def → delayed bleeding + umbilical stump bleeding 

vit K dependent factors

• Factors: II, VII, IX, X

• protein C and S

• require vit K → y-carboxylation (Ca binding)

• def → prolongen PT first (FVII shortens half-life)

natural anti-coagulants (3 + what happens when def)

• antithrombin → inhib thrombin + FXa

• protein C/S system → inact factors Va + VIIIa

• TFPI → inhib TF-VIIa complex 

• def → high risk of thrombosis 

fibrinolysis

• plasmin breaks down fibrin

• tPA act plasminogen → plasmin

• D-dimer = fibrin degradation product

fibrinolysis inhibi = 2

• PAI-1 → inhibits tPA

• α2-antiplasmin → inactivates plasmin

• Balance prevents premature clot lysis.

d-dimer

• Marker of cross-linked fibrin breakdown.

• Elevated in:

  • thrombosis

  • DIC

  • infection

  • inflammation.

• High sensitivity, low specificity.

PT = prothrombin time → measures, sensitive to, when prolonged

• Measures extrinsic + common pathway.

• Sensitive to factor VII, X, V, II, I.

• Prolonged in

  • warfarin use

  • vitamin K deficiency

  • liver disease.

aPTT

• measures intr + common pw 

• prolonged in

  • HA or HB

  • heparin therapy

  • lupus anticoag

thrombin time = TT

• measures conversion of fibrinogen → fibrin

• prolonged in

  • low fibrinogen 

  • dysfibrinogenemia

  • hepa use

HA → which factor, inheritance, wha tis prolonged, clinical

• FVIII defi

• X linked recessive 

• prolonged aPTT

• joint/muscle bleeds 

HB

• FIX defi

• same as HA

hemophilia treatment

• Factor replacement → VIII or IX

• Desmopressin → mild hemophilia A (↑ FVIII release).

• Emicizumab → FVIII bridging.

vWF:Ag

• measures amount of vWF protein → not function

vWF:Act (or GPIbM binding assay)

• Measures functional activity of vWF.

• Indicates GPIb binding capacity

vWF:CB (Collagen-binding assay)

• Measures ability of vWF to bind to collagen.

• Reduced in some type 2 variants.

RIPA = (Ristocetin-induced platelet aggregation)

• Tests interaction between vWF and GPIb.

• Hyperresponsive in type 2B.

• Hyporesponsive in type

  • 2A / 2M

  • type 1.

FVIII:C

• Factor VIII coagulant activity.

• Function test for FVIII.

• Low in VWD (especially type 3, 2N).

vWF multimers

Loss of high-molecular-weight multimers = type 2A or 2B.

DDAVP response

• Measures rise of vWF + FVIII after desmopressin.

• Good in type 1 VWD.

• Contraindicated in type 2B.

thrombocytosis → count, causes (2)

• platelet count > 450 × 10⁹ /L

• causes 

  • primary → myeloproliferative neoplasma (ET, PV)

  • secondary → inflamma, infection, iron def

immune thrombocytopenia = ITP → what happens, tests?

• AI destruc of platelets → IgG

• Normal coagulation tests.

• Treatment: steroids, IVIG, TPO agonists.

Drug-induced platelet dysfunction (4)

• Aspirin: irreversible COX-1 inhibition → ↓ TxA2

• NSAIDs: reversible COX inhibition

• P2Y12 inhibitors: clopidogrel, ticagrelor → ↓ ADP signaling

• GPIIb/IIIa inhibitors: tirofiban, eptifibatide → block aggregation

Uremic platelet dysfunction

• seen in renal failure 

• Impaired adhesion + aggregation.

• Normal platelet count.

• Treat with dialysis or DDAVP.

Virchovs triad

• hemodynamic changes/stasis

• hypercoagulability

• endothelial injury or dysfunction

art thrombosis → how is the clot, leads to, treat with?

• Platelet-rich clot (“white clot”).

• Triggered by atherosclerotic plaque rupture.

• Leads to MI or stroke.

• Treated with antiplatelet drugs.

ven thrombosis = DVT → rich with, caused by, risk, treat with

• Fibrin + RBC-rich clot (“red clot”).

• Caused by stasis + hypercoagulability.

• Risk: PE.

• Treated with anticoagulants.

pulmonary embolism = PE

• Thrombus from DVT travels to pulmonary arteries.

• Symptoms: dyspnea, chest pain, tachycardia.

• Life-threatening.

cancer associated hypercoag

• tumor releases → procoag factors → TF, cytokines

• act endothelium + platelets

• incr thrombin generation

trouesseau syndrome

• Migratory thrombophlebitis in cancer.

• Often pancreatic or gastric cancer.

heparin/LMWH

• anticoag

• act antithrombin = AT

• inhib Xa + IIa (thrombin)

• LMWH → more selective to inhib IIa

warfarin

• anticoag

• inhib vit K epoxide reductase = VKORC1

• reduces y-carboxylation of factors → II, VII, IX, X

• monitor by INR 

DOAC

• anticoag

• Xa inhib → rivarox-, apix-, endoxaban

• IIa inhib → dabigatran

clinical clues primary hemostasis disorders

• petechiae !

• small superficial bruises

• epitaxis + gum bleeds !

• menorrhagia

clinical clues secundary hemostasis disorders

• large ecchymoses

• joint bleeds

• muscle bleeds

PA-dependent plasmin generation

• tPA from endothelial cells

• Converts plasminogen → plasmin

• Requires fibrin as cofactor

• tPA only active when bound to fibrin

• Prevents systemic fibrinolysis

• Plasminogen + tPA bind fibrin → plasmin formed locally

uPA-dependent plasmin generation

• Activator = sc-uPA (pro-uPA)

• Produced by endothelium, macrophages, epithelial cells

• Plasmin activates sc-uPA → tc-uPA (urokinase)

• tc-uPA converts plasminogen → plasmin

• Works with or without fibrin

• Positive feedback loop: plasmin ↑ → more tc-uPA ↑ → more plasmin

Plasminogen Activator Inhibitor-1 (PAI-1) → prod by, inhibits what, prevents…

• Produced by: endothelium, platelets, monocytes

• Inhibits tPA and tc-uPA

• Strong, rapid inhibitor

• Prevents fibrinolysis outside thrombus

Regulation of plasmin generation

• Depends on tPA and uPA activity

• Controlled by inhibitors (PAI-1, α2-antiplasmin)

• Requires fibrin surface for localized activation

• Prevents systemic plasmin activity

α2-antiplasmin → produced by, inhibits what, prevents…

• Produced by liver

• Inhibits free plasmin in circulation

• Prevents plasmin diffusion away from clot

• Ensures fibrinolysis only occurs locally

How fibrin regulates plasmin generation

• Fibrin provides binding sites for tPA + plasminogen

• Only fibrin-bound tPA is active

• Lysine residues on fibrin allow plasminogen binding

• Thrombin-activated TAFI removes lysines → reduces binding → slows fibrinolysis

AFI (Thrombin-Activatable Fibrinolysis Inhibitor)

• Circulates as inactive pro-carboxypeptidase

• Activated by thrombin → TAFIa

• Removes lysine residues from fibrin

• Reduces plasminogen + tPA binding

• Slows fibrinolysis and stabilizes clot

Venous thrombosis:

• Low flow (stasis), fibrin-rich clots

• Risk = DVT → pulmonary embolism

• Treatment: heparin, VKA, DOACs

• Clot removed by endogenous fibrinolysis

Arterial thrombosis:

• Triggered by diseased vessel wall (atherosclerosis)

• Platelet-rich, high shear

• Can cause obstruction → ischemia/infarct

• Cleared by endogenous + pharmacological fibrinolysis

artheroslerose step 1

• endothelial dysfunction → LDL enterst → oxidation

• oxLDL = toxic → triggers endothelial activation

• increase adhesion mol expression

• recruits monocytes 

artherosclerose step 2

• monocyte migrate into intima → macrophage

• mp engulfs oxLDL → foam cells → cant exit

• fatty streak → first visible lesion 

arthero step 3

• SMC migrate from media → intima 

  • prolif in response to injury

  • produce collagen + elastin 

• form fibrous cap over lipid core

art step 4

• foam cell death → necrotic lipid core

• collagen rich fibrious cap forms → plaque 

art step 5

• aging + cell death → Ca depostion 

• artery becomes stiff = sclerosis

• vessel can remodel

  • outward → no symp

  • inward → stenosis = reduced flow

art step 6

• plaque rupture

• infla cells release proteases

• colla breakdown > synthese

• fib cap thins → rupture → expose thrombo core

kanker pt en VTE risico

• hoogste risico in eerste 3 maanden

• daarna zakt het af

Trousseau-syndroom

• Migrerende tromboflebitis + kanker

• Caused by tumor-derived TF + procoagulant microparticles

• Typisch bij pancreas- of longkanker

Waarom maakt kanker een hypercoagulabele staat?

• Tumorcellen produceren TF + procoagulante microparticles

• NETs activeren stolling

• Cytokines ↑ trombinevorming

• Compressie van vaten door tumor

• Chemo + anti-angiogene therapie ↑ stolling

• Immobilisatie

Mechanisme: hoe activeert tumor de stollingscascade?

• Tumorcellen → expressie TF → activeert extrinsieke pathway

• Trombine → fibrinevorming → trombus

• Extra versterkt door platelets + microparticles

Behandeling VTE bij kanker

• 1e keuze: LMWH voor 6 maanden

• Daarna: eventueel VKA (minder gebruikt tegenwoordig)

• DOAC’s vaak ook effectief, maar risico op GI-bloeding bij GI-tumoren

• Behandeling gericht op preventie PE

Biologische risicofactoren voor VTE bij kanker

• Microparticles (MP)

• NETs (Neutrophil Extracellular Traps)

• ↑ stollingsfactoren

• Circulating tumor cells

• Interactie tumorcellen–bloedplaatjes–leukocyten

• Tumorcompressie van vaten

Wat zijn microparticles (MP) en waarom geven ze trombose?

• Kleine membraanfragmenten van tumorcellen of bloedcellen

• Dragen TF, fosfatidylserine, eiwitten, micro-RNA

• Kunnen direct stolling activeren

• MP–TF hoogte = biomarker voor tumoractiviteit + tromboserisico

Diagnostische methoden om microparticles te detecteren 3

• FACS: telt MPs >500 nm

• Functional assays: clotting time verkort bij meer TF

• ELISA: detecteert TF op MPs in plasma

Wat zijn NETs en hoe veroorzaken ze kanker-gerelateerde trombose? → act facort

• Neutrophil Extracellular Traps = web van DNA + proteïnen

• Normaal: vallen pathogenen

• In kanker: NET–DNA activeert factor XII → trombine

• NETs binden plaatjes → versterken trombus

• NETs verhogen visco-elasticiteit van stolsel

Welke genetische mutaties verhogen tromboserisico bij kanker?

• Tumorcellen met KRAS activatie → ↑ TF expressie

• Ook ↑ microparticle shedding

Rol van IHC (immunohistochemie) in tromboserisico bij kanker

• Detecteert TF-expressie, REG4, SerpinA1

• REG4/SerpinA1: markers voor hoog tromboserisico

• Kan helpen bij risicostratificatie

Hoe bevordert fibrine metastasering bij CRC + VTE?

• Meer fibrine = dichter extravasaal netwerk

• Hypoxie stimuleert EMT en angiogenese

• Hypoxie → HIF-1α → invasie + metastase

• Tumorcellen gebruiken fibrine als matrix voor migratie

Hoe activeren stollingsfactoren tumorcellen? → welke facotren activeren wat, wat is gevolg van die activitatie

• Factor IIa (trombine) en Xa binden PAR-receptoren

• PAR activatie →

  • ↑ angiogenese

  • ↑ invasie

  • ↑ metastase

  • verhoogde tumoroverleving