Background

Cardiovascular diseases are the leading cause of death worldwide, with coronary artery disease, cerebrovascular disease, and peripheral vascular disease being the most prevalent. These conditions are often managed using vascular stents or grafts, but thrombosis remains a significant challenge, contributing to the failure of these devices. As a result, patients with stents or grafts typically require blood thinners, which can cause serious side effects. Therefore, minimizing thrombogenicity is a critical focus for both commercially available stents and grafts and the development of new vascular biomaterials.

The surfaces of vascular biomaterials interact with blood components to initiate coagulation through the intrinsic clotting cascade. To help minimize the risk of thrombogenesis, anticoagulants such as sodium citrate, sodium ethylenediaminetetraacetic acid (EDTA), and sodium heparin are often used. These anticoagulants are capable of chelating calcium ions, reducing the concentration of free calcium in the blood, and thus inhibiting multiple clotting factors such as factors II, IX, X, XI, and XII, which depend on calcium for their activation. Blood coagulation factor XII (FXII) activates when it binds to artificial, negatively charged surfaces such as biomaterials. This activation transforms FXII into the enzyme factor XIIa, which activates FXI, leading to the generation of thrombin [1]. FXII activation can occur in vitro and has been shown to have a role in intravascular thrombus formation, which makes it ideal for our research.

The surfaces of vascular biomaterials interact with blood components, triggering coagulation through the intrinsic clotting cascade. To reduce the risk of thrombogenesis, anticoagulants such as sodium citrate, sodium ethylenediaminetetraacetic acid (EDTA), and sodium heparin are commonly employed. These anticoagulants chelate calcium ions, thereby lowering the concentration of free calcium in the blood and inhibiting calcium-dependent clotting factors, including factors II, IX, X, XI, and XII. Blood coagulation factor XII (FXII) is particularly relevant, as it is activated upon binding to artificial, negatively charged surfaces, such as those of vascular stents or grafts, leading to the generation of thrombin [1]. In addition to FXII activation, soluble fibrinogen is converted into insoluble fibrin during thrombin generation. This conversion can be detected through changes in light absorbance within a blood plasma sample. The formation of fibrin also promotes platelet aggregation, further contributing to the development of a hemostatic clot [2].

This protocol monitors the production of fibrin by measuring the change in opacity of PPP or PRP over time, using the absorbance measured at a wavelength of 405 nm. This method provides insights into the thrombogenicity of various biomaterials and evaluates the influence of biological factors such as anticoagulants on blood plasma.

The use of platelet-rich plasma (PRP) vs. platelet-poor plasma (PPP) relies on the type of information being tested. PRP has a higher-than-normal amount of platelets, whereas PPP has barely any platelets at all. PRP contains several growth factors that support tissue regeneration, such as epidermal growth factor (EGF), transforming growth factor-beta (TGF-b), platelet-derived growth factor (PDGF), and insulin-like growth factor 1 (IGF-1) [3]. PRP also contains more calcium-binding proteins and clotting factors, whereas PPP contains more fibrinogen and lacks platelet-specific components. PRP may be more helpful in regenerative studies, while PPP may be useful in studies where platelets complicate the analysis of results. PRP clots faster than PRP due to the presence of platelets.

Sodium citrate, EDTA, and heparin are all capable of chelating calcium ions; however, they are not equally effective in this method. While heparin inhibits blood coagulation factor IV (calcium ions), it also inhibits other clotting factors that will not be reactivated by the recalcification of blood. As such, we expect this protocol to be less effective when using heparin vs. other anticoagulants. Other similar methods [4] include a partial thromboplastin time (PTT) test, which is used clinically to evaluate blood disorders based on recalcifying blood and measuring the time it takes to clot. Our protocol can be used for clinical applications as well; however, it is targeted toward the evaluation and improvement of biomaterials. Because it evaluates various anticoagulants for their efficacy in creating a thrombus after recalcification, our protocol could also be used to evaluate the efficacy of various anticoagulants in calcium chelation. In addition, this protocol can be used to evaluate the activation of FXII on various biomaterials.