REVISED MODELS OF THE CONTACT ACTIVATION BLOOD PLASMA-COAGULATION CASCADE

Open Access
Author:
Pitakjakpipop, Harit
Graduate Program:
Materials Science and Engineering
Degree:
Master of Science
Document Type:
Master Thesis
Date of Defense:
July 31, 2017
Committee Members:
  • James Hansell Adair, Thesis Advisor
Keywords:
  • Blood coagulation
  • FactorXII
  • FactorXI
  • Autoactivation Surface
  • FactorXIIa
Abstract:
About 25 percent of Americans are currently living with some form of cardiovascular disease with approximately 800,000 deaths each year. Millions of cardiovascular devices are used each year in the US. However, all biomaterials eventually lead to some form of thrombosis. Therefore, surface engineering of biomaterials with improved hemocompatibility is an imperative, given the widespread global need for cardiovascular devices. Investigating the biochemical reaction of blood factor proteins in contact with an activation surface is the major purpose of these studies. Research summarized in this thesis focuses on contact activation of FXI and FXII in buffer and blood plasma, a process usually referred to as autoactivation. In Chapters 2 and 3, recent hematology literature combined with new theoretical and experimental results from our laboratory offers an updated version of blood plasma coagulation cascade biochemistry in vitro for use in the study of blood-biomaterial interactions. A revised version of the coagulation cascade is proposed that emphasizes a known role for FXI in controlling the time required to complete plasma coagulation after activation with hydrophilic procoagulant surfaces such as glass. Central to this revision is a “thrombin (FIIa) amplification route” that has been generally recognized in existing models of in vivo plasma coagulation cascade biochemistry but has been afforded the proper emphasis as a pivotal feedback loop that activates FXI in vitro, in a way similar to that known to occur in vivo. Thus, a connection between in vitro and in vivo coagulation is drawn. We propose that this thrombin amplification route is catalyzed by the substantial amount of FIIa generated in coagulating plasma that persists in serum prepared from coagulated plasma. This revision (Fig. 3.4) further includes an updated version (Fig. 3.5) of the contact activation phase mediated by surface activation of the Hageman factor known as FXII. In Chapter 4, a contact activation of FXII in buffer solutions (i.e. in absence of plasma proteins) with hydrophilic and silanized-glass activators spanning the observed range of water wettability (hydrophilic to hydrophobic), shows no evidence based on high-resolution electrophoresis evaluating proteolytic cleavage of FXII into αFXIIa or βFXIIa. The autoactivation mixture contains only a single-chain protein with a molecular weight of ~80 kDa, confirming the previous finding of Oscar Runoff of a single-chain activated form of FXII that he called ‘HFea’. Functional assays have shown that these autoactivation products exhibit procoagulant potential (protease activity inducing clotting of blood) or amidolytic potential (cleaves amino bonds in s-2302 chromogen but do not cause coagulation of plasma) or both amidolytic potential and procoagulant potential. Some of these proteins also have the remarkable potential to ‘suppress autoactivation’ (i.e. suppress creation of enzymes with procoagulant potential). It is thus hypothesized that autoactivation of FXII in the absence of plasma proteins generates not just a single type of activated conformer, as suggested by previous researchers, but rather an ensemble of conformer products with collective activity that varies with activator surface energy used in contact activation of FXII. Furthermore, reaction of αFXIIa with FXII in buffer solution does not produce additional αFXIIa by the putative autoamplification reaction FXII + FXIIa  2FXIIa as has been proposed in past literature to account for the discrepancy between chromogenic and plasma-coagulation assays for αFXIIa in buffer solution. Instead, net procoagulant activity measured directly by plasma-coagulation assays decreases systematically with increasing FXII solution concentration. Under the same reaction conditions, chromogenic assay reveals that net amidolytic activity increases with increasing FXII solution concentration. Thus, although autoamplification does not occur, it appears that there is some form of “FXII self-reaction” that influences the products of αFXIIa reaction with FXII. Electrophoretic measurements indicate that no proteolytic cleavage takes in this reaction leading us to conclude that change in activity is most likely due to change(s) in FXII conformation (with related change in enzyme activity).