NOVEL THERAPEUTIC STRATEGIES

TO REGULATE THROMBOSIS AND HEMOSTASIS

Research

Welcome to the Kastrup Lab. We are a group of biochemists and engineers working to gain a deeper upstanding of how blood clotting is regulated and can be controlled therapeutically. Severe hemorrhage is a major cause of death, particularly among young people, while thrombosis is a major contributor to death and disability with age. We use proteins and cells naturally involved in blood clotting to develop novel technologies for addressing unmet needs in controlling severe bleeding and preventing unwanted clotting.

The research in our lab is focused on these questions:

  1. What new technologies are needed to halt bleeding in the most severe cases of hemorrhage?
  2. Can platelets be genetically modified to enhance their hemostatic activity, or targeted to treat Alzheimer’s disease?
  3. Can clot stability be altered to treat hemorrhage or thrombosis?
  4. Do soil and microbes in wounds influence bleeding?

We use a variety of biochemical assays, RNA biotechnology, biomaterial synthesis, imaging, patient blood, microfluidics and human blood, and small and large animal models to answer these questions.

Propelling therapeutics deep into wounds

Severe hemorrhage can be triggered by trauma, surgery, or childbirth, and requires timely treatment with blood products and hemostatic agents to stop the blood loss. The most severe cases of hemorrhage are characterized by a widespread failure of the body’s blood clotting system, and bleeds can rapidly get out of control. Hemostatic agents are often applied topically into wounds to stop bleeding, but current agents are often ineffective in severe hemorrhage because they are flushed out of the wound by high blood flow.

To address this challenge, the we have developed microparticles that propel upstream through blood and deliver therapeutics deep into wounds. These microparticles, loaded with a powerful pro-coagulant enzyme, thrombin, successfully reduced blood loss and mortality in small and large animal models of bleeding in trauma, surgery and endoscopy. These hemostatic products are being translated in collaboration with commercial and military partners. We are currently testing the ability of these microparticles to deliver antimicrobials into wounds to prevent infection during injury.

Enhancing the function of platelets and megakaryocytes

Platelets are small, anucleate blood cells essential for hemostasis. In response to vascular damage, platelets activate and aggregate to form a plug to seal the wound and prevent blood loss. As part of this process, activated platelets release molecules that promote clot formation in a localized manner. 

Platelet transfusions are used to stop or prevent bleeding, but these are often ineffective during the most severe cases of hemorrhage, when platelets become dysfunctional and their contribution to hemostasis is lost. We are developing ways to enhance the natural procoagulant function of platelets to improve their hemostatic efficacy.

We have developed an approach to load nucleic acids and proteins into platelets using lipid nanoparticles (LNPs). We have demonstrated that platelets loaded with thrombin formed clots significantly faster and stronger than un-modified platelets, even in blood plasma from patients with clotting defects. We are now exploring whether we can further extend the therapeutic function of platelets by using LNPs to deliver mRNA to the platelets. We are also investigating whether LNPs can deliver therapeutics to megakaryocytes, platelet precursor cells found within the lungs and bone marrow, to modify platelet function.

Controlling clot stability to regulate hemostasis

During hemorrhage, fibrin is covalently crosslinked to itself and the vessel wall to form a meshwork that seals the wound. If this meshwork is not stably linked to the vessel wall, the wound is not effectively sealed, leading to re-bleeding. This process is mediated by coagulation factor XIIIa (FXIIIa), a transglutaminase enzyme found in platelets and blood. We are currently exploring whether supplementing the blood with additional FXIIIa is a feasible strategy to strengthen clots that form during severe bleeding. Furthermore, our lab has shown that the enzymes responsible for degrading fibrin, tissue plasminogen activator (tPA) and plasmin, also degrade FXIIIa. Since tPA is used clinically as clot-busting drug in patients with thrombosis, we are investigating whether the activity of FXIIIa is altered in patients that have received this treatment.

Thrombosis is also treated with anticoagulants that target events upstream of fibrin crosslinking to prevent additional clots from forming in the patient. However, these carry the risk of unwanted bleeding. We hypothesized that a safer approach is instead to inhibit the crosslinking of fibrin, which would allow an initial plug to form and seal the wound, but also increase the susceptibility of the clot to degradation. To test this hypothesis, we are exploring whether knockdown of FXIIIa, and proteins that regulate its activity, are alternative therapies for thrombosis.

Investigating new mechanisms for controlling clotting

Coagulation is controlled by a complex network of reactions that were first described over 50 years ago. Research over the decades has led to a better understanding of the biochemistry of the coagulation network, yet questions still remain about aspects of blood clotting. Humans deficient in coagulation factor XII (FXII) do not appear to bleed spontaneously or excessively after trauma, leading to a debate over its role in blood clotting. FXII is activated by silicates, a major component of soil, to clot blood in vitro. Interestingly, FXII is been conserved in land-dwelling animals, but not in animals that do not live on soil, such as marine mammals. We therefore asked the question, is soil a physiological activator of FXII? If so, then FXII may have roles in blood clotting that have never before been characterized, and we are exploring these. (Helen, Vancouver Aquarium. Cropped photo by Analise Zocher.)

Neurodegenerative diseases have also been linked to defective clotting. For example, amyloid beta (Aβ), a key protein associated with Alzheimer’s disease, interacts with coagulation proteins and can accumulate along the inside of blood vessels during cerebral amyloid angiopathy. We recently showed that FXIIIa can crosslink Aβ to itself and to fibrinogen to make clots stiffer. We are currently exploring the physiological significance of this interaction to determine whether Aβ is a normal component of blood clots in the brain, or if FXIIIa contributes to the pathology of cerebral amyloid angiopathy. As platelets are an abundant source of Aβ precursor protein, we are also investigating the physiological role of Aβ precursor protein in platelets.

Aspects of the above projects are currently funded by CIHR, MSFHR and NSERC.