## 4 Discussion  
Static tests to evaluate thrombosis are relatively simple and yield more consistent results. However, device thrombogenicity related to blood flow and surface topography cannot be evaluated and limit the prediction of the long-term outcomes of implantable medical devices. In addition, distinguishing subtle differences in thrombosis between biomaterials may not be possible. This study confirmed the blood coagulation potential in various animal species based on the ASTM F2382-18:PTT, a test method standardized and approved by the FDA, and identified the differences in reactivity of different species for complement. Furthermore, a hemocompatibility evaluation model was designed, and the information required to interpret the results was obtained. The PTT assay is a modification of the activated partial thromboplastin time (APTT) assay. The APTT assay includes active agents such as kaolin, celite, or ellagic acid [17], which must be avoided when evaluating the effects of blood contact devices or device materials because it interrupts coagulation caused by the materials or device [18]. Unlike the APTT assay, the PTT assay evaluates whether the test material acts as an activator using reagents (rabbit brain cephalin) without activating substances such as kaolin, celite, or ellagic acid. The time it takes for citrate plasma exposed to the test material when exposed to phospholipid particles and calcium chloride suspension to form a clot is measured. Shortening the PTT after contact with a substance indicates activation of the intrinsic coagulation pathway of blood coagulation, whereas heparin and other anticoagulants induce prolonged PTTs [1; 15]. Despite variations in coagulation factors, such as F-IX, F-X, and F-VII, across species, the coagulation pathway may essentially be similar among mammals [19; 20; 21]. Many studies have been conducted using different animal species for blood coagulation, and the degree of coagulation has been reported to differ depending on the animal species [5; 6; 22]. The applicability of using animal blood instead of human blood with the test blood of PTT assay was examined to determine the blood clotting potential in various animal species. PTT changes were observed with one negative and two positive control candidate materials as the test material and plasma from rabbits, porcine, rhesus monkeys, and cynomolgus monkeys. The absolute value of the PTT reaction in the plasma of rabbits, porcine, and primates that reacted with the material showed slight differences among animals. However, the normalized values--61% for rabbits, 66% for pigs, 63% for rhesus monkeys, and 65% for cynomolgus monkeys--did not show any statistically significant differences. The evaluation of thrombosis using PTT demonstrated the possibility of replacing human blood if an appropriate animal model is selected. The complement is activated by the surfaces of external materials, including blood-contacting medical devices, and usually occurs in the early stages [23; 24]. The amount of complement proteins in the blood can be evaluated using ELISA assays, and the complement proteins that can be used in ELISA assays include, but are not limited to, C3a, C5a,  
Figure 4: Comparison of complement activity in human and porcine sera reacted with the CVF. (**A**) Effects of test substances on SC5b-9 production. (**B**) Effects of test substances on C3a production. The mean \(\pm\) SD values of the three independent experiments are shown. and SC5b9; C3a is considered a general indicator of complement activation, and SC5b-9 is generally considered a marker representing the full extent of complement activation [25; 26; 27]. An increase in complement components indicates that the complement system is activated. Only complement components in plasma can be measured, and measuring complement components that attach to the material surface is difficult. Commercially available ELISA kits are species-specific, and a suitable control group needs to be included for comparison.