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Molecular Biology

Coagulopathies

Welcome to the world of hemostasis, the body’s incredibly elegant system for stopping bleeding. You can think of it as a biological construction project. When there’s a break in a blood vessel wall, a complex cascade of clotting factors (proteins) is activated to rapidly build a fibrin “dam”—a blood clot—to plug the hole. Just as important is the clean-up crew, the anticoagulant proteins that dissolve the clot once the vessel is healed and prevent the dam from getting dangerously large

Normally, this system is in perfect balance. But what if there’s an inherited genetic quirk that tips the scale in favor of clotting? This leads to a state of hypercoagulability, or thrombophilia—an increased tendency to form dangerous and unnecessary blood clots in the veins (venous thromboembolism, or VTE) or arteries

Our job in the molecular lab isn’t to diagnose an active clot; that’s done with imaging. Our role is to identify the underlying hereditary risk factors by reading the patient’s DNA. This is particularly important for patients who have had a clot at a young age, in an unusual location, or have a strong family history of clotting

The Big Players in Hereditary Thrombophilia

We’re looking for specific, common spelling mistakes in the DNA called single nucleotide polymorphisms (SNPs). These are single-letter changes in the genetic code that alter the function of key clotting proteins. The two most important culprits we test for are Factor V Leiden and the Prothrombin gene mutation

Factor V Leiden: The “Resistant” Clotting Factor

This is, by far, the most common inherited cause of thrombophilia, especially in Caucasian populations

  • The Analogy: Think of the clotting cascade as a team of workers building that fibrin dam. Activated Protein C (APC) is the safety inspector whose job is to tell a specific worker, Factor V, to stop working so the dam doesn’t get too big
  • The Molecular Defect: The Factor V Leiden mutation is a specific SNP (a G-to-A change) in the F5 gene. This results in a single amino acid change (Arginine is replaced by Glutamine at position 506). This single change happens to be right where the “safety inspector” (APC) binds to Factor V to shut it down
  • The Consequence: The mutated Factor V Leiden protein is resistant to being inactivated by APC. The “safety inspector” can’t do its job. As a result, this hyperactive Factor V worker keeps building the clot for longer than it should, dramatically increasing the risk of thrombosis
  • How We Test: Because we’re looking for one specific SNP, we use targeted PCR-based methods. Real-time PCR with melt-curve analysis is a common and elegant approach. We can clearly distinguish the DNA melting patterns of a normal individual (homozygous wild-type), a carrier (heterozygous), and a rare affected individual (homozygous mutant)

Prothrombin G20210A: The “Over-Producer”

This is the second most common inherited clotting disorder. It works by a completely different mechanism

  • The Analogy: If Factor V is a worker, think of Prothrombin (also called Factor II) as the raw material, the “concrete mix” for the dam
  • The Molecular Defect: The Prothrombin G20210A mutation is a SNP (a G-to-A change) located in a non-coding part of the F2 gene called the 3’ untranslated region (3’ UTR). This region helps control how much protein is made from the gene blueprint
  • The Consequence: This mutation makes the prothrombin mRNA transcript more stable, preventing it from being broken down as quickly. The cell’s protein-making machinery can therefore read this stable transcript over and over, leading to an overproduction of prothrombin protein. With too much “concrete mix” floating around, the balance is tipped towards clot formation
  • How We Test: Just like with Factor V Leiden, this is a single SNP, making it perfect for targeted PCR assays

MTHFR: The “Retired” Player

You will undoubtedly see orders for, and hear discussions about, variants in a gene called MTHFR (methylenetetrahydrofolate reductase). This gene is involved in folate metabolism, and mutations can lead to elevated levels of an amino acid called homocysteine, which was historically linked to thrombosis

  • The Molecular Defect: Two common variants are often tested: C677T and A1298C
  • The Current Clinical View: This is a critical teaching point. Decades of research have shown that while MTHFR variants are common, their association with venous thromboembolism is very weak to non-existent. Major clinical practice guidelines, including those from the American College of Medical Genetics and Genomics (ACMG), now strongly recommend against testing for MTHFR variants as part of a thrombophilia workup. It does not change patient management and is not considered a reliable risk factor for VTE
  • Our Role as MLSs: It’s our job to understand the evidence. While we will run the test if a physician orders it, we must also be aware that its clinical utility for thrombophilia is no longer supported. This is a great example of how molecular diagnostics evolves with scientific evidence

Key Terms

  • Thrombophilia: An inherited or acquired condition that increases the tendency to form abnormal blood clots (thrombosis). Also known as hypercoagulability
  • Thrombosis: The formation of a blood clot inside a blood vessel, obstructing the flow of blood through the circulatory system
  • Hemostasis: The physiological process that causes bleeding to stop, meaning to keep blood within a damaged vessel. It is the first stage of wound healing
  • Factor V Leiden: A specific mutation in the F5 gene that makes the Factor V protein resistant to inactivation by Activated Protein C, leading to an increased risk of venous thrombosis
  • Prothrombin G20210A: A specific mutation in the 3’ untranslated region of the prothrombin (F2) gene that leads to increased levels of prothrombin protein, predisposing an individual to thrombosis
  • Activated Protein C (APC) Resistance: The functional consequence of the Factor V Leiden mutation, where the mutated Factor V protein cannot be efficiently “turned off” by its natural inhibitor, Activated Protein C
  • Single Nucleotide Polymorphism (SNP): A common type of genetic variation involving a change at a single position in a DNA sequence. Factor V Leiden and Prothrombin G20210A are classic examples of pathogenic SNPs