Partially Reduced Graphene Oxide as a Promoter of Thrombin Activity and Fibrinogen Clot Formation

Approximately 40% of trauma-related deaths worldwide are due to bleeding, highlighting the need to create a topical dressing that would enhance the rapid clotting of deep cuts and/or wounds. Reduced graphene oxide has been studied extensively for its electronic properties; partially reduced graphene oxide (pRGO) contains fewer oxygen-containing functional groups than graphene oxide (GO), which increases its electrical conductivity but maintains some of the functional groups that still keep it water-soluble. Previously it was shown that pRGO enhances the ability of the enzyme microbial transglutaminase to crosslink gelatin. Since thrombin is an enzyme that catalyzes fibrinogen clot formation, we sought to examine whether pRGO would also enhance thrombin’s enzymatic activity and speed the formation of clots.<br/>A 1 mg/mL GO solution was reduced with sodium borohydride (NaBH4) to obtain 10, 12, 15, 20 and 25 mM reductions of pRGO. Fibrinogen (4 mg/ml) was clotted with thrombin (1 u/ml) and a thromboelastograph quantified the viscoelastic properties of the sample including the R value, which is the time it takes to begin forming the clot to an amplitude of 2mm; the angle of the tangent to the clotting curve, which indicates the rate of clot formation; the time it takes to fully form the clot; and the maximum amplitude (MA), which shows the strength of the clot.<br/>The addition of pRGO significantly enhances thrombin’s activity in all aspects. Although GO improves the initial clotting rate (indicated by the tangent of the angle) by 17%, pRGO boosts it by 63%. Whereas GO reduces overall clotting time by 27%, pRGO reduces the overall clotting time by 60% . However, compared to the control, pRGO improves the maximum amplitude of the clot by 71%, while GO decreases it by 6%.<br/>Testing the various reduction degrees of prGO demonstrated that 20mM exerts the most significant enhancement, increasing the initial clotting rate by 147%, enhancing the clot strength by 154%, and decreasing the overall clotting time by 60%. To derive the optimal reduction degree for a pRGO promoter, the mean of the extremums of the values as far as angle to the curve, maximum amplitude, and total clotting time for each pRGO reduction was determined to be 19 mM.<br/>Halving the amount of thrombin in the control to 0.5 u/mL results in roughly half the initial clotting rate as compared to the full 1 u/mL thrombin control. However, halving the amount of thrombin while adding 20 mM pRGO nearly triples the initial clotting rate of halved thrombin and even speeds the clotting rate 56% faster than the full 1 u/mL thrombin control.<br/>Increasing the amount of pRGO while keeping the amount of thrombin constant at 0.75 u/mL was shown to progressively increase the angle of the tangent (thereby decreasing the initial clotting time) but eventually reached a maximum plateau.<br/>Hence, partially reduced graphene oxide shows significant promise as a promoter of thrombin’s activity to clot fibrinogen, with far-reaching implications for healthcare as prGO’s ability to enhance thrombin’s clotting efficiency could be a major breakthrough in managing severe wounds and improving survival rates for trauma patients.