1. Rapid sulfopropyl-disk chromatographic purification of bovine and human thrombin
H C Whinna, F C Church Anal Biochem . 1986 Aug 15;157(1):77-83. doi: 10.1016/0003-2697(86)90198-3.
Thrombin, from either a crude commercial preparation (bovine) or a prothrombin activation mixture (human), was purified by sulfopropyl-disk chromatography to homogeneity in a rapid and convenient single-step procedure. The yield of both proteinases was greater than 85%. Purified bovine and human thrombin had sp act of 2500 and 3000 "NIH" units/mg, respectively. Human thrombin was more reactive than bovine thrombin with these active site-directed probes: phenylmethylsulfonyl fluoride, dansyl-Glu-Gly-Arg-chloromethylketone, and human heparin cofactor II. The sulfopropyl-disk chromatographic method is a useful and rapid technique to prepare milligram quantities of highly purified bovine and human thrombin.
2. Selective anticoagulation with active site blocked factor IXa in synthetic patch vascular repair results in decreased blood loss and operative time
A M Schmidt, R Nowygrod, D M Stern, E A Rose, M C Oz, J D Madigan, T B Spanier ASAIO J . 1997 Sep-Oct;43(5):M526-30.
Heparin has been the mainstay of anti thrombic therapy in arterial repair procedures. With increasing use of synthetic patch angioplasty (polytetrafluoroethylene [PTFE] or Dacron, Medical Products, Flagstaff, AZ) to improve long-term patency and limit aneurysmal dilation, however, the use of heparin has been associated with excessive needle hole bleeding, resulting in time delay in the operating room to achieve hemostasis, as well as clinically significant blood loss. Because of the multiple sites of action of heparin in the coagulation cascade, both intravascular (desired effect) and extravascular (untoward side effect) hemostasis are impaired. The authors therefore tested the hypothesis that selective inhibition of intravascular coagulation, without significant impairment of extravascular hemostasis, would prevent clotting intraluminally while preserving hemostasis at the suture line of the patch graft. The unique position of factor IX/IXa in the coagulation cascade renders its inhibition an ideal target in this setting. The authors prepared active site blocked factor IXa (IXai) using dansyl-Glu-Gly-Arg chloromethylketone, and tested this hypothesis in a New Zealand rabbit aortotomy model with PTFE patch closure using either heparin (25 i.u./kg; n = 16) or IXai (300 micrograms/kg; n = 21). The infrarenal aorta was identified and isolated, the anti coagulant infused, aortic cross clamp placed, and aortotomy repaired with a 2 x 6 mm PTFE patch. After cross-clamp removal, blood loss was measured and time to hemostasis was recorded. Compared with heparin, IXai resulted in significantly reduce blood loss (6.97 +/- 4.4 g vs 2.72 +/- 2.51 g, respectively, p < 0.008), and time to hemostasis (2.94 +/- 0.77 min vs 2.0 +/- 0.63 min, respectively, p < 0.003). To assess long-term patency and thrombosis, 12 rabbits (given heparin; n = 6 and IXai; n = 6) were observed for up to 2 months post-operatively. No differences were observed between rabbits treated with heparin or IXai; 100% of the grafts were patent with no differences in degree of intimal hyperplasia by histologic analysis. Together, these data suggest that use of IXai in PTFE vascular repair will safely allow realization of the benefits of long-term patency and decreased aneurysmal dilatation, while eliminating the intraoperative morbidity of needle hole bleeding.