Blood Coagulation and Thrombolytic Therapies

Introduction

  • Bill Atkins, enzymologist focusing on drug metabolism, particularly enzymes.

  • Research involves biophysical and biochemical techniques.

  • Aims to bridge understanding between small molecule drugs and larger protein drugs, focusing on older protein drugs developed during the early days of protein engineering.

  • Will highlight key concepts for quizzes/tests.

Small Molecules vs. Protein Drugs

  • Small Molecules:

    • Molecular weight: < 1000 Da.

    • Made by organic synthesis.

    • Goal: Oral administration for better patient compliance.

  • Protein Drugs:

    • Molecular weight: > 10 kDa (e.g., antibodies are larger).

    • Made biologically (cell culture, bacteria).

    • Administered intravenously or subcutaneously.

    • No oral protein drugs available yet.

  • Shared Concepts:

    • Optimize pharmacodynamic (PD) and pharmacokinetic (PK) properties.

    • Manipulate structure to:

      • Slow down clearance.

      • Increase half-life.

      • Change distribution.

      • Minimize toxicity.

    • For protein drugs, this involves manipulating protein sequence, removing/modifying domains.

  • Important Note:

    • This is a simplification; other modalities exist (peptides, nucleic acids, hybrid molecules).

Blood Coagulation: A Historical Perspective

  • Historical Significance:

    • Ancient cultures (Egyptians) used bleeding in ceremonies.

    • Greeks believed in bloodletting for therapeutic reasons, balancing "humors". Mapped anatomical locations for safe blood withdrawal.

    • 19th century Europe: Bloodletting as routine health practice.

  • Modern Alternative Medicine:

    • Leeches used for migraine headaches, varicose veins.

      • Leeches secrete local anesthetics and anticoagulants.

    • Mechanical leeches:

      • Devices with needles to extract blood, inject anesthetics and anticoagulants.

      • Used in microsurgery to prevent clot formation in small vasculature.

Overview of Blood Coagulation

  • Homeostatic Mechanisms:

    • Continuous surveillance for wounds/clots.

    • Balance between clot formation and prevention.

  • Relevance:

    • Control/prevention of strokes and heart attacks.

    • Rapid clot degradation after heart attack/stroke onset improves prognosis.

  • Process:

    • Blood vessel damage leads to release of signaling molecules.

    • Platelets activate, aggregate, form a platelet plug.

    • Activated platelets recruit red blood cells.

    • Fibrin forms, stabilizing the clot.

    • Key Point: Fibrin is essential for clot stabilization; a primary target in thrombolytic therapy.

Blood Coagulation Pathways

  • Intrinsic and extrinsic pathways converge to activate factor X.

  • Zymogens: Inactive proteases (most blood factors are zymogens).

    • Factor X (inactive) → Factor Xa (active protease)

    • Prothrombin (Factor II) → Thrombin (Factor IIa)

    • Thrombin cleaves fibrinogen → Fibrin

    • AT3 (antithrombin III) inhibits thrombin and other blood factors.

  • Direct Oral Anticoagulants: Inhibit Factor Xa or thrombin (e.g., apixaban).

    • Used prophylactically in patients post-heart attack/stroke.

  • Key Point: Conversion of fibrinogen to fibrin is critical.

Hirudine: A Thrombin Inhibitor

  • Leeches secrete hirudine to prevent blood clots during feeding.

  • Hirudine binds to thrombin, inhibiting its activity.

  • Recombinant hirudine is available for therapeutic use (not first-line).

Tissue Plasminogen Activator (tPA)

  • More common thrombolytic strategy: degrade fibrin at clot site.

  • tPA secreted by cells lining vasculature.

  • Fates of tPA:

    • Liver uptake (limits duration of action).

    • Binding to PAI1 (plasminogen activator inhibitor-1), which inhibits tPA.

    • Binding to fibrin (desired outcome).

  • tPA binds to fibrin, cleaving plasminogen → plasmin.

  • Plasmin degrades fibrin, breaking down the clot.

    • Plasminogen→tPAPlasminPlasminogen \xrightarrow{tPA} Plasmin

  • Issue: tPA also binds to fibrinogen, leading to systemic activation and potential hemorrhaging.

  • Goal: High fibrin specificity over fibrinogen.

  • Natural tPA has modest specificity.

Desired tPA Characteristics

  • Not taken up by the liver.

  • Not inhibited by PAI1.

  • Longer half-life (single bolus injection).

  • Non-antigenic.

  • High fibrin specificity.

Thrombolytic Drugs: Streptokinase

  • Streptokinase:

    • Old drug, still widely used due to lower cost.

    • Bacterial protein (streptococcal strains).

    • NOT an enzyme: Binds and activates plasminogen, causing a conformational change without cleavage.

    • Mechanism: Prevents blood clot formation, aiding bacterial invasion.

    • Antigenic: Can cause immune responses.

    • Short half-life: Requires constant IV drip (~3 minutes).

Thrombolytic Drugs: Alteplase (Recombinant Human tPA)

  • Alteplase (recombinant human tPA):

    • Complex protein (~520 amino acids), color-coded by domain.

      • Finger domain.

      • Epidermal growth factor (EGF) domain.

      • Two Kringle domains.

      • Serine protease domain (catalytic activity).

    • Expressed in mammalian cell cultures, glycosylated.

    • EGF and Kringle 1 domains: Responsible for receptor binding and clearance.

    • Kringle 2: Binds PAI-1 (inhibitor).

    • Short half-life: Requires constant IV drip (5-6 minutes).

    • Drawback: Binds to fibrinogen in circulation causing activation and hemorrhaging.

Thrombolytic Drugs: Tenecteplase (Modified tPA)

  • Tenecteplase (modified tPA):

    • Full-length tPA with site-specific mutations based on structure-function studies.

    • Mutations:

      • Glycosylation sites (103 & 117) mutated in Kringle 1 to prolong half-life and reduce hepatic uptake.

      • Positive charge patch (arginines/lysines) in Kringle 2 converted to alanines to reduce PAI-1 binding.

    • Advantage:

      • Significantly increased duration of action (25 minutes), allowing single bolus injection.

      • Potentially used more frequently than alteplase in the future due to convenience.

Thrombolytic Drugs: Reteplase (Truncated tPA)

  • Reteplase (truncated tPA):

    • Cut off finger and growth factor domains, as well as Kringle 1.

    • Lower hepatic uptake and clearance due to loss of glycosylation sites.

    • Significantly decreased affinity for fibrin.

    • Has a longer duration of action, as long as thirteen to fifteen minutes.

    • Advantage:

      • Potentially diffuses into the clot better (speculative).

Thrombolytic Drugs: Lanoteplase (Truncated tPA)

  • Lanoteplase (truncated tPA):

    • Cut off the finger domain and the fibronectin domain.

    • Contains a single point mutation in Kringle-one at the site of glycosylation.

    • Slightly decreased susceptibility to hepatic uptake.

    • Half life that ranges from fifteen to forty minutes.

    • Advantage:

      • Can potentially be used as a single injection

Comparing Thrombolytic Drugs

  • Alteplase derivatives (alteplase, reteplase, lanoteplase):

    • Not immunogenic.

  • Streptokinase:

    • Immunogenic.

  • Key Questions:

    • Which drugs have better fibrin specificity?

    • Which have longer durations of action?

  • Logic Behind Drug Design:

    • Target glycosylation or PAI1 binding to modify hepatic uptake and inhibition.

    • Goal: Single-injection drugs with high fibrin specificity.

Thrombolytic Drugs: Desmotoplase (Vampire Bat-Inspired)

  • Desmoteplase (vampire bat-inspired):

    • Inspired by tPA variants in vampire bats that feed on mammals.

    • Vampire bats that feed on mammals have deletions of kringle two.

    • Kringle 2 deletion leads to PAI1 insensitivity.

    • High fibrin specificity.

    • Long half-life (hours).

    • Clinical trials discontinued (reason unclear).

Conclusion

  • Thrombolytics are useful for heart attacks and strokes.

  • Protein structures can be manipulated like small molecules to change pharmacogenetic/pharmacological properties via protein engineering.