Mod 1: Blood Disorders

Hemostasis Requirements (RLCS)

Rapid

Controlled

Site-Specific

Limited

Hemostasis: Rapid

Blood loss prevention should occur as soon as wound occurs

Hemostasis: Controlled

Regulated response

Hemostasis: Site-Specific

Only occur at wound

Hemostasis: Limited

Blood clot dissolve when not needed

Procoagulant State

Promote wound healing and prevent blood loss

Endothelial damage, platelet activation, coagulation activation

Anticoagulant State

Prevent thrombosis

Anticoagulant factors, fibrinolytic factors, healed endothelium

Thrombosis

Thrombus (blood clot) formation

Endotheliums

Blood and vascular

Blood Components: Liquid

Plasma

Plasma Function

Transport proteins (coagulation factors), hormones, electrolytes, organic nutrients, waste products

Blood Components: Cellular

Platelets, erythrocytes, leukocytes

Platelet Function

Initiate hemostasis

Platelet: Quiescent State

Intact endothelium

Vascular Endothelium

Endothelial monolayer

Vascular Endothelium Function

Produce factors preventing hemostasis

Separate blood from procoagulant factors

Subendothelium

Connective tissue beneath endothelium containing ECM, smooth muscle, and procoagulant factors

Disrupting Vascular Endothelium

Initiate hemostasis

Blood contact with procoagulant factors = Platelet plug formation

Primary Hemostasis

Endothelial disruption expose subendothelium

Vasoconstriction

Platelet plug formation

Secondary Hemostasis

Coagulation cascade produce fibrin and hemostatic plug

Tertiary Hemostasis

Fibrinolysis (degrade fibrin and hemostatic plug)

Regulatory mechanisms

Primary Hemostasis: Vasoconstriction

Reduce blood loss

Damaged endothelial cells release endothelin

Primary Hemostasis: Platelet Plug Formation

von Willebrand Factor (vWF) bind collegen in subendothelium

Circulating platelet vWF receptors bind vWF

Platelets activate, change shape, and release granules to recruit more platelets

Secondary Hemostasis: Coagulation Cascade

Form fibrin and hemostatic plug

Secondary Hemostasis: Hemostatic Plug

Cross-linked fibrin stabilize platelet plug, trap erythrocytes and leukocytes

Strengthen platelets

Secondary Hemostasis: Coagulation Factor Activation

Inactive zymogen form + Co-factor + Activated enzyme → Active enzyme form

Secondary Hemostasis: Procoagulant Surface

Activated phospholipid bilayer and endothelial cells required for hemostatic plug formation

Coagulation Factor: Activated FX (FXa)

Catalyze prothrombin → Thrombin

Thrombin

Soluble Fibrinogen → Insoluble Fibrin (clot)

FX → FXa

Intrinsic or extrinsic tenase complex

FXa: Intrinsic Tenase Complex

FXIIa → FXIa → FIXa + FVIIIa + FX → FXa

Amplify coagulation response

Increase fibrin production

FXa: Extrinsic Tenase Complex

Tissue Factor + FVIIa + FX → FXa

Generate small amounts of fibrin

Intrinsic Pathway: Positive Regulation

Thrombin amplify intrinsic pathway factors

FXI, FVIIIa, FXIIIIa

Tertiary Hemostasis: Fibrinolysis

Break down cross-linked fibrin → Degradation products

Control clot size and spread

Remove clot after vessel repair

Fibrinolysis: Plasminogen Activators

uPA and tPA

Convert plasminogen zymogen → Active plasmin → Break down fibrin clot

Fibrinolysis Pathway: Thrombin

Fibrin degradation products inhibit thrombin → Inhibit fibrinogen to fibrin conversion

No hemostatic plug reformation

Fibrinolysis Pathway: Plasminogen Activator Inhibitor (PAI)

Inhibit uPA and tPA

Fibrinolysis Pathway Dysfunction

Uncontrolled hemostatic plug block blood vessel

Primary Hemostasis: Negative Regulation

Endothelium-derived inhibitory mechanisms prevent excess platelet adhesion, activation, and aggregation

NO, prostacyclin, CD39

Negative Regulation: Nitric Oxide (NO)

Gas secreted by healthy endothelium to prevent platelet adhesion and aggregation

Negative Regulation: Prostacyclin

Lipid secreted by healthy endothelium to prevent platelet adhesion and aggregation

Negative Regulation: CD39

Transmembrane ATPase expressed by healthy endothelium

Catalyze ATP dephosphorylation to prevent platelet activation

Anticoagulants

Tissue factor pathway inhibitor, antithrombin, protein C

Anticoagulant: Tissue Factor Pathway Inhibitor (TFPI)

Inhibit extrinsic pathway

Produced by endothelial cells and megakaryocytes

Anticoagulant: Antithrombin

Interact with heparin on endothelial cell surfaces

Inactivate coagulation factors (FVII, FIX, X, XI, XII, thrombin)

Produced by hepatocytes

Heparin

Liver and tissue compound inhibiting coagulation

Anticoagulant: Protein C

Zymogen activated by thrombin + thrombomodulin

Requires protein S cofactor to mediate proteolytic inactivation (FVIIIa, FVa)

Measuring Blood Proteins

Centrifuge blood sample to separate cellular components and plasma

Tube coated with anticoagulant (EDTA) to prevent serum collection

Plasma vs Serum

Plasma: Contain proteins

Serum: Plasma without clotting factors (in clot)

Hemophilia

Inherited bleeding diathesis (susceptibility to bleeding)

Defective coagulation system

No intrinsic pathway activation to produce sufficient fibrin

Hemophilia A

FVIII protein deficiency

Decreased FVIIIa = Decreased FIXa efficiency = Decreased FX activation

Hemophilia B

FIX protein deficiency

Decreased FIXa = Decreased FX activation

Diagnosing Hemophilia

FVIII/FIX levels < 50%

Mild Hemophilia

FVIII/FIX levels 5-50% of normal

Moderate Hemophilia

FVIII/FIX levels 1-5% of normal

Severe Hemophilia

FVIII/FIX levels < 1% of normal

Hemophilia Etiology

Monogenic disease

Mutation in F8 (A) or F9 (B) gene on X chromosome

Point mutation most common

Hemophilia Inheritance

X-linked recessive pattern

Hemophilia Inheritance: Males

1 gene copy cause hemophilia

More likely affected

Hemophilia Inheritance: Females

2 gene copies cause hemophilia

Carriers: 1 gene copy

Unlikely to be affected

F8 Gene

186 kb

26 exons

F9 Gene

33\.5 kb

8 exons

Hemophilia: Detect DNA Mutations

1. Extract DNA from peripheral blood mononuclear cells
2. Isolate DNA
3. Amplify exons with PCR
4. Next generation sequencing

Detect DNA Mutations: Next Generation Sequencing

Compare patient DNA to reference

Severe Hemophilia A Causes

F8 gene intron inversions

Intron 22 (more common), intron 1

Severe Hemophilia A: Next Generation Sequencing

Cannot use

Normal DNA sequence with inverted axons

Sequencing cannot tell the difference between inversion and normal reference

Severe Hemophilia A Diagnosis

1. Intron 22 Inversion
2. Intron 1 Inversion
3. PCR for point mutations

Hemophilia Complications

Spontaneous bleeding and provoked bleeding

Hemophilia Complications: Spontaneous Bleeding

Localized to joints and soft tissue

Hemophilia Complications: Provoked Bleeding

From trauma or surgical procedures

Hemophilia Treatments

Maintain FVIII/FIX levels

Protein replacement therapy, PEGylation, Emicizumab, Fitusiran, gene replacement therapy

Protein Replacement Therapy

Infuse FVIII or FIX into patient

Protein Replacement Therapy Challenge: Delivery

Intravenous administration

Short half life (frequent IV)

* FVIII: 12 hours
* FIX: 24 hours

Protein Replacement Therapy Challenge: Anti-Drug Antibody

Anti-FVIII antibodies decrease treatment effectiveness

Protein Replacement Therapy Challenge: Cost

Difficult access for low SES

Protein Replacement Therapy Challenge: Infectious Disease

Pathogens pass through plasma (HIV, Hep A/B/C)

Clotting factors screened for pathogens and treated with chemical and physical inactivators

Hemophilia Treatment: PEGylation

Add polyethylene glycol polymer (PEG) chains to block interactions with FVIII clearance receptors

Prolong endogenous factor half-life

Hemophilia Treatment: Emicizumab

Bispecific antibody binding FIXa and FX

Partial FVIII mimetic molecule

Improve intrinsic tenase complex catalytic activity

Increase FXa formation

Emicizumab: Therapeutic Benefits

Subcutaneous administration

Longer half-life

Active in anti-FVIII antibody presence

Low immunogenicity

Hemophilia Treatment: Fitusiran

Short interfering RNA (siRNA)

Inhibit antithrombin (anticoagulant) production

Rebalance procoagulant and anticoagulant factors

Hemophilia Treatment: Gene Replacement Therapy

Normal gene introduced

Endogenous protein production

Mutation repair, non-viral gene transfer, cell-based gene therapy, viral gene transfer

Gene Replacement Therapy: Mutation Repair

Using CRISPR-Cas 9 gene editing

Gene Replacement Therapy: Non-Viral Gene Transfer

Using targeted nanoparticles

Gene Replacement Therapy: Cell-Based Gene Therapy

Using embryonic stem cells

Gene Replacement Therapy: Viral Gene Therapy

Using adeno-associated virus (AAV), retrovirus, lentivirus

Viral Gene Transfer 1: DNA + AAV

Insert therapeutic DNA into viral vector

Viral Gene Transfer 2: Target Cell

Deliver virus + DNA to target cell

Viral Gene Transfer 3: Protein

Target cell makes therapeutic protein

FVIII/FIX Transgene Delivery 1: AAV Virus Genome

4\.7kb single stranded DNA

2 inverted terminal repeats (ITR)

2 genes (Rep and Cap)

FVIII/FIX Transgene Delivery 2: AAV Vector Genome

Remove Rep and Cap genes

Insert promoter and FVIII transgene insertion

FVIII/FIX Transgene Delivery 3: AAV Vector Particle

AAV vector genome in caspid

FVIII/FIX Transgene Delivery 4: Patient Administration

Intravenous

Hepatotropic AAV serotype/variation taken up by liver cell nuclei

Protein produced from transgene

Thrombophilia

Thrombus formation

Reduced anticoagulant proteins or increased procoagulant proteins

Early Thrombus: Ischemia/Hypoxia

Ischemia: Restrict blood flow

Hypoxia: O2 deprivation

Late Thrombus: Infarction

Tissue necrosis from long-term O2 deprivation

Embolisation

Thrombus break and travel into lungs

Cause death

Venous Thrombosis

Thrombi form at valves

Superficial Thrombophlebitis

In veins close to skin (palpable)

Localized pain

Low embolization risk

Venous Thromboembolism (VTE)

Deep vein thrombosis (DVT) and pulmonary embolism (PE)

DVT

In deep veins

Proximal (above knee) more likely to embolize than distal (below knee) DVTs