Abstract
Treatment of thrombosis typically involves the administration of coagulation inhibitors that must be carefully monitored and balanced so as to reduce unwanted coagulation (thrombosis) while maintaining normal or near-normal hemostasis. This balancing is necessary since the anticoagulants used alter enzymatic activities that are involved in both processes. This therapeutic strategy is based entirely on the view that thrombosis occurs by the same general pathways as normal hemostasis. While the enzymatic cascade of blood coagulation is well described and well accepted, numerous other minor reactions have been shown to occur in vitro but have not been examined in great detail due to the belief that they do not occur significantly during normal coagulation in vivo. We postulate that in certain pathological environments some of these minor procoagulant reactions may in fact become significant and lead to thrombogenic situations. If true, this could potentially allow novel targets for anticoagulation to be identified. In addition, the inhibition of these abnormal reactions could attenuate pathological coagulation whilst having limited or no effect on normal hemostatic reactions. One candidate reaction is the proteolysis of factor VIII (fVIII) by the factor VIIa-tissue factor (fVIIa-TF) complex, which results in a mixture of active and inactive fVIII molecules. We have previously shown that this reaction occurs in vitro using purified plasma components and in situ in a plasma-based system. Both of these systems produce a low level of fVIII activation with sustained (albeit low) fVIIIa activity. While it remains possible that this reaction is important in early hemostasis the elevated levels of TF in many pathological situations raises the possibility that this reaction may be more pronounced under certain circumstances in disease states. Examination of the importance of this reaction in vivo is an extremely important issue, but very difficult to address due to the inability to ascertain if fVIII activity or fragments found in vivo derive from fVIIa-TF proteolysis or proteolysis by other enzymes such as thrombin, factor Xa, or activated protein C. With this in mind we have developed an antibody reagent that can specifically detect a fVIII fragment that is a unique product of fVIII proteolysis by the fVIIa-TF complex. This antibody detects only fVIIa-TF proteolyzed fVIII (fVIII cleaved at Arg336) and its major product (α-fragment) on Western blots but not intact (unactivated) fVIII or thrombin-activated fVIII. Using this antibody we screened samples of pulmonary lavage and pleural fluid from normal patients as well as patients with acute respiratory distress syndrome, interstitial lung disease, pneumonia and lung cancer—all of which have associated procoagulant pathologies. Sandwich ELISAs of patient samples showed variably elevated levels of α-fragment (from 100 – 2000 pM) compared to normal controls (~5 pM). Western blots of lavage samples confirmed the presence of α-fragment in samples as well as the elevated levels compared to normals. These data strongly support the notion that alternative “abnormal” coagulation products can be and are generated in vivo in certain pathological settings. The data are also strongly suggestive that the fVIIa-TF complex is the most likely source of fVIII α-fragment. Although it remains unclear if fVIII α-fragment is one of the causative agents in the procoagulant pathologies of these disorders or merely an indicator of the abnormal procoagulant state, its presence in vivo indicates that the role of abnormal coagulation reactions should be further investigated.
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