The Regulatory Function Of Amino Acid Region 473-487 Of Prothrombin During Coagulation

Background In the United States, every thirty nine seconds an individual dies from complications from Cardiovascular Diseases. Persistent thrombus formation at the genesis of these diseases, such as stroke and other coagulation disorders, has no full model to date. Intrinsically blood clots are produced due to excessive/unnecessary thrombin formation, which leads to the conversion of fibrinogen to fibrin. As a result the regulation of thrombin formation is critical in controlling clot generation. Upon vasculature damage, the proteolytic conversion of prothrombin (Pro) to thrombin compatible to rates of survival is catalyzed by the prothrombinase complex composed of the enzyme, factor Xa (fXa), the cofactor, factor Va (fVa), assembled on a phospholipid membrane in the presence of calcium ions. Although fXa is capable of activating Pro through the initial cleavage at Arg271 followed by the cleavage at Arg320 (pre2 pathway), it would take approximately six months to form a clot. However, the incorporation of fVa into prothrombinase results in a five-fold increase in the catalytic efficiency of fXa for thrombin generation and the order of cleavages reversed (meizo pathway). Thus the timely arrest of unwarranted bleeding is due to the assembly of prothrombinase at the site of injury. Inevitably the presence and absence of fVa dictates the pathway of Pro activation and previous studies have suggested that fXa interacts with Pro within amino acid region 473-487 in a fVa-dependent manner. Aim To evaluate the role amino acid region 473-487 of Pro has in coagulation. Methods A recombinant Pro molecule with the region 473-487 was deleted (rProΔ473-487) using site-directed mutagenesis. Methotrexate was used for selection to stably transfect BHK-21 cells with rProΔ473-487 and wild-type Pro (rProWT). The two recombinant molecules were purified according to a well-established protocol and, at the last step, Fast Performance Liquid Chromatography was used equipped with a strong anionic Mono-Q 5/50 column. Properly carboxylated rProΔ473-487 and rProWT was isolated and removed from the column by utilizing a calcium gradient. Subsequently Pro deficient plasma was used to assess the molecules clotting activities on a Diagnostica Stago STart® 4 Hemostasis Analyzer. Gel electrophoresis was used to evaluate both recombinant molecules and their ability to generate active thrombin by either the multifaceted prothrombinase or fXa alone. Further studies were then performed using generated recombinant thrombin from the recombinant Pro molecules to investigate in their ability to activate procofactors V (fV) & VIII (fVIII). Results The investigation into the Activated Partial Thromboplastin Time [APTT] revealed clotting activity for human Pro and rProWT to be comparable, whereas rProΔ473-487 was substantially limited in the process of forming a fibrin clot. Next gel electrophoresis and scanning densitometry indicated the consumption of rProΔ473-487 by prothrombinase and subsequent thrombin formation was decreased 24-fold when compared to rProWT. In contrast membrane-bound fXa alone, in the absence of fVa, exhibited a 6-fold increase in the rate of initial cleavage Arg271 and subsequent activation of rProΔ473-487. Both recombinant Pro molecules demonstrated a similar cleavage pattern of activation equivalent with human Pro suggesting no structural alterations took place in rProΔ473-487following the mutation. Furthermore, generated human thrombin and recombinant wild-type thrombin were found to activate fV and fVIII within five minutes while the recombinant mutant thrombin was impeded in the activation process out to three hours. Conclusion Overall the data demonstrate that amino acid sequence 473-487 of Pro plays a preeminent role in 1) timely activation of Pro at initial cleavage Arg320 by prothrombinase, and 2) suitable macromolecular procofactor activation. Thus there is incisive rationale why no major mutations have been identified in this dynamic region which would be problematic for inherent physiological hemostasis. Disclosures: No relevant conflicts of interest to declare.