Factor IXa

Abstract: Platelet activation and blood coagulation are complementary, mutually dependent processes in haemostasis and thrombosis. Platelets interact with several coagulation factors, while the coagulation product thrombin is a potent platelet-activating agonist. Activated platelets come in a procoagulant state after a prolonged elevation in cytosolic [Ca2+]i. Such platelets, e. g. when adhering to collagen via glycoprotein VI, expose phosphatidylserine (PS) at their outer surface and produce (PS-exposing) membrane blebs and microvesicles. Inhibition of aminophospholipid translocase and activation of phospholipid scramblase mediate the exposure of PS, whereas calpain-mediated protein cleavage leads to membrane blebbing and vesiculation. Surface-exposed PS strongly propagates the coagulation process by facilitating the assembly and activation of tenase and prothrombinase complexes. Factor IXa and platelet-bound factor Va support these activities. In addition, platelets can support the initiation phase of coagulation by providing binding sites for prothrombin and factor XI. They thereby take over the initiating role of tissue factor and factor VIIa in coagulation activation.

Abstract: Many therapeutic agents are associated with adverse effects in patients. Anticoagulants can engender acute complications such as significant bleeding that increases patient morbidity and mortality. Antidote control provides the safest means to regulate drug action. For this reason, despite its known limitations and toxicities, heparin use remains high because it is the only anticoagulant that can be controlled by an antidote, the polypeptide protamine. To date, no generalizable strategy for developing drug-antidote pairs has been described. We investigated whether drug-antidote pairs could be rationally designed by taking advantage of properties inherent to nucleic acids to make antidote-controlled anticoagulant agents. Here we show that protein-binding oligonucleotides (aptamers) against coagulation factor IXa are potent anticoagulants. We also show that oligonucleotides complementary to these aptamers can act as antidotes capable of efficiently reversing the activity of these new anticoagulants in plasma from healthy volunteers and from patients who cannot tolerate heparin. This generalizable strategy for rationally designing a drug-antidote pair thus opens up the way for developing safer regulatable therapeutics.

Abstract: Factor VIII has been purified approximately 300000-fold from bovine plasma by ammonium sulfate fractionation, glycine precipitation, DEAE-Sephadex column chromatography, sulfate--Sepharose column chromatography, Sephadex G-200 gel filtration, and factor X--Sepharose column chromatography. The highly purified preparation migrated as a triplet on sodium dodecyl sulfate/urea--polyacrylamide gel electrophoresis with apparent molecular weights of 93000, 88000, and 85000. The coagulant activity of the purified preparations was inhibited by antibodies raised in rabbits against either the purified factor VIII protein or a preparation of factor VIII/von Willebrand factor. Antibodies to the purified protein also inhibited the coagulant activity of factor VIII/von Willebrand factor preparations. The purified factor VIII contained no platelet-aggregating activity, as measured in human platelet-rich plasma. The purified preparation of factor VIII was required for the activation of factor X in the presence of factor IXa, calcium, and phospholipid. It was activated about 30-fold by thrombin or factor Xa plus calcium and phospholipid, and each of these reactions was accompanied by a change in the sodium dodecyl sulfate/urea--polyacrylamide gel electrophoresis pattern of the protein. Factor VIII was rapidly inactivated by bovine-activated protein C in a reaction requiring calcium and phospholipid. This reaction was also associated with a change in the sodium dodecyl sulfate/urea--polyacrylamide gel electrophoresis pattern of the highly purified protein. These experiments involving three highly specific serine proteases support the conclusion that the triplet observed on polyacrylamide gels is factor VIII.

Abstract: Hemophilia A is a bleeding disorder resulting from coagulation factor VIII (FVIII) deficiency. Exogenously provided FVIII effectively reduces bleeding complications in patients with severe hemophilia A. In approximately 30% of such patients, however, the 'foreignness' of the FVIII molecule causes them to develop inhibitory antibodies against FVIII (inhibitors), precluding FVIII treatment in this set of patients. Moreover, the poor pharmacokinetics of FVIII, attributed to low subcutaneous bioavailability and a short half-life of 0.5 d, necessitates frequent intravenous injections. To overcome these drawbacks, we generated a humanized bispecific antibody to factor IXa (FIXa) and factor X (FX), termed hBS23, that places these two factors into spatially appropriate positions and mimics the cofactor function of FVIII. hBS23 exerted coagulation activity in FVIII-deficient plasma, even in the presence of inhibitors, and showed in vivo hemostatic activity in a nonhuman primate model of acquired hemophilia A. Notably, hBS23 had high subcutaneous bioavailability and a 2-week half-life and would not be expected to elicit the development of FVIII-specific inhibitory antibodies, as its molecular structure, and hence antigenicity, differs from that of FVIII. A long-acting, subcutaneously injectable agent that is unaffected by the presence of inhibitors could markedly reduce the burden of care for the treatment of hemophilia A.