Loma Linda University

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Danilo Boskovic, PhD
Assistant Professor, Basic Sciences
School of Medicine
Assistant Professor, Earth and Biological Sciences
School of Medicine
Member, Faculty of Graduate Studies
Publications    Scholarly Journals--Published
  • Plank,M.S., Boskovic,D.S., Sowers,L.C. and Angeles,D.M. (2008.08.01) Biochemical Markers of Neonatal Hypoxia.  Pediatr.Health 2(4):485-501. ( 8/2008 )
  • Calderon,T.C., Wu,W., Rawson,R.A., Sakala,E.P., Sowers,L.C., Boskovic,D.S. and Angeles,D.M. (2008.07.01)  Effect of Mode of Birth on Purine and Malondialdehyde in Umbilical Arterial Plasma in Normal Term Newborns.  J.Perinatol. 28(7):475-481. ( 7/2008 )
  • Danilo S. Boskovic, Thomas Troxler and Sriram Krishnaswamy. (2004.05.14) Active Site-independent Recognition of Substrates and Product by Bovine Prothrombinase. A Fluorescence Resonance Energy Transfer Study.   J.Biol.Chem. 279(20):20786-20793. ( 5/2004 ) Link...
    The conversion of prothrombin to thrombin is catalyzed by prothrombinase, an enzyme complex composed of the serine proteinase factor Xa and a cofactor protein, factor Va, assembled on membranes. Kinetic studies indicate that interactions with extended macromolecular recognition sites (exosites) rather than the active site of prothrombinase are the principal determinants of binding affinity for substrate or product. We now provide a model-independent evaluation of such ideas by physical studies of the interaction of substrate derivatives and product with prothrombinase. The enzyme complex was assembled using Xa modified with a fluorescent peptidyl chloromethyl ketone to irreversibly occlude the active site. Binding was inferred by prethrombin 2-dependent perturbations in the fluorescence of Oregon Green488 at the active site of prothrombinase. Active site-independent binding was also unequivocally established by fluorescence resonance energy transfer between 2,6-dansyl tethered to the active site of Xa and eosin tethered to the active sites of either thrombin or meizothrombin des fragment 1. Comparable interprobe distances obtained from these measurements suggest that substrate and product interact equivalently with the enzyme. Competition established the ability of a range of substrate or product derivatives to bind in a mutually exclusive fashion to prothrombinase. Equilibrium dissociation constants obtained for the active site-independent binding of prothrombin, prethrombin 2, meizothrombin des fragment 1 and thrombin to prothrombinase were comparable with their affinities inferred from kinetic studies using active enzyme. Our findings directly establish that binding affinity is principally determined by the exosite-mediated interaction of either the substrate, both possible intermediates, or product with prothrombinase. A single type of exosite binding interaction evidently drives affinity and binding specificity through the stepwise reactions necessary for the two cleavage reactions of prothrombin activation and product release.
  • Danilo S. Boskovic, Laszlo S. Bajzar and Michael E. Nesheim. (2001.08.03) Channeling During Prothrombin Activation.  J. Biol.Chem. 276(31):28686-28693. ( 8/2001 ) Link...
    The plasma zymogen prothrombin (II) is converted to the clotting enzyme thrombin (IIa) by two prothrombinase-catalyzed proteolytic cleavages. Thus, two intermediates, meizothrombin (mIIa) and prethrombin-2 (P2), are possible on the reaction pathway. Measurements of the time courses of II, mIIa, P2, and IIa suggested a channeling phenomenon, whereby a portion of the II is converted directly to IIa without free mIIa and P2 as obligatory intermediates. Evidence for this was that the maximum rate of IIa formation preceded the maximum in the level of either intermediate. In addition, analysis of the data according to a model that included two parallel pathways through mIIa and P2 indicated that about 40% of the II consumed did not yield free mIIa or P2. Further studies were carried out in which II was continuously infused in a reactor at a constant rate. Under these conditions II, mIIa, and P2 reached constant steady-state levels, and IIa was produced at a constant rate, equal to that of II infusion. During the steady state, traces of II, mIIa, and P2 were introduced as radiolabels. Time courses of isotope consumption were first order, thus allowing the rates of consumption of II, mIIa, and P2 to be calculated. Under these conditions the rate of II consumption equaled the rate of IIa formation. Rates of consumption of the free intermediates, however, were only 22 (mIIa) and 15% (P2), respectively, of the rate of thrombin formation. Thus, both the time course experiments and the steady-state experiments indicate that an appreciable fraction of II is channeled directly to IIa without proceeding through the free intermediates mIIa and P2.
  • Danilo S. Boskovic and Sriram Krishnaswamy. (2000.12.08) Exosite Binding Tethers the Macromolecular Substrate to the Protheombinase Complex and Directs Cleavage at Two Spatially Distinct Sites.  J.Biol.Chem. 275(49):38561-38570. ( 12/2000 ) Link...
    The prothrombinase complex, composed of the proteinase, factor Xa, bound to factor Va on membranes, catalyzes thrombin formation by the specific and ordered proteolysis of prothrombin at Arg323-Ile324, followed by cleavage at Arg274-Thr275. We have used a fluorescent derivative of meizothrombin des fragment 1 (mIIaDF1) as a substrate analog to assess the mechanism of substrate recognition in the second half-reaction of bovine prothrombin activation. Cleavage of mIIaDF1 exhibits pseudo-first order kinetics regardless of the substrate concentration relative to Km. This phenomenon arises from competitive product inhibition by thrombin, which binds to prothrombinase with exactly the same affinity as mIIaDF1. As thrombin is known to bind to an exosite on prothrombinase, initial interactions at an exosite likely play a role in the enzyme-substrate interaction. Occupation of the active site of prothrombinase by a reversible inhibitor does not exclude the binding of mIIaDF1 to the enzyme. Specific recognition of mIIaDF1 is achieved through an initial bimolecular reaction with an enzymic exosite, followed by an active site docking step in an intramolecular reaction prior to bond cleavage. By alternate substrate studies, we have resolved the contributions of the individual binding steps to substrate affinity and catalysis. This pathway for substrate binding is identical to that previously determined with a substrate analog for the first half-reaction of prothrombin activation. We show that differences in the observed kinetic constants for the two cleavage reactions arise entirely from differences in the inferred equilibrium constant for the intramolecular binding step that permits elements surrounding the scissile bond to dock at the active site of prothrombinase. Therefore, substrate specificity is achieved by binding interactions with an enzymic exosite that tethers the protein substrate to prothrombinase and directs cleavage at two spatially distinct scissile bonds.
  • Danilo S. Boskovic, Alan R. Giles, and Michael E. Nesheim. (1990.06.25) Studies of the Role of Factor Va in the Factor Xa-catalyzed Activation of Prothrombin, Fragment 1.2-Prethrombin-2, and Dansyl-L-glutamyl-glycyl-L-arginine-meizothrombin in the Absence of Phospholipid.  J.Biol.Chem. 265(18):10497-10505. ( 6/1990 ) Link...
    In order to specifically evaluate the role of Factor Va in the prothrombinase complex, studies of the activation of prothrombin, Fragment l?2-prethrombin-2, and active-site-blocked meizothrombin were carried out, both in the absence of phospholipid and at concentrations of substrates and Factor Va sufficient to approach saturation in all components. Km values were independent of Factor Va concentrations, whereas kcat(apparent) values approached saturation with respect to Factor Va concentrations. The three respective substrates exhibited the following parameters of kinetics (Km, µM; kcat, 1/s at saturating [Factor Va]): prothrombin (9.0 ± 0.4; 31 ± 1); Fragment l?2-prethrombin-2 (5.4 ± 0.4; 13 ± 2); and meizothrombin (3.6 ± 0.3; 51 ± 5). Models of kinetics were constructed to interpret the results, and two of these were formally consistent with experimental results. Both models indicated that the variation of kcat(app) with concentrations of Factor Va reflects the formation of a Factor Va-Factor Xa binary complex. Analysis of kinetics indicated Kd values for this interaction of 1.3 ± 0.1, 3.0 ± 0.5, and 1.0 ± 0.1 µM for the three respective substrates. The models differed in the interpretation of Km. One indicated that Km reflects a binary interaction between Factor Xa and prothrombin, whereas the other indicated a binary interaction between Factor Va and prothrombin. Both indicated that two of the three possible binary interactions between the three components would be reflected in Km and kcat values but not the third. To distinguish these models, the binary interactions were studied by extrinsic fluorescence (Va*Xa), light-scattering (Factor Va*prothrombin), and competition kinetics (Xa*II). The first two interactions were detected and were characterized by Kd values of 2.7 ± 0.1 µM (Va*Xa) and 8.8 ± 0.8 µM (Factor Va*prothrombin). No active-site-dependent interaction between prothrombin and Factor Xa could be detected in the absence of Factor Va. The results of these studies suggest that Factor Va interacts with both Factor Xa and prothrombin and effectively presents one to the other in the formation of a ternary enzyme-substrate-cofactor complex. In addition, a comparison of the parameters of kinetics of conversion of prothrombin and its intermediates indicates that meizothrombin is the major intermediate of prothrombin activation in the absence, as well as in the presence of phospholipid.
  Books and Chapters
  • Calderon,T.C., Boskovic,D.S., Sowers,L.S. and Angeles,D.M. (2008)  Use of Purines and Xanthine Oxidase as Markers of Neonatal Hypoxia-Ischemia. Chapter 8.  In: Advancements in Neurological Research. Edited by Zhang,J.H. Research Signpost, Kerala, India, pp:209-227. ( 8/2008 )
  • Michael E. Nesheim, Russell P.Tracy, Paula B. Tracy, Danilo S. Boskovic and Kenneth G. Mann. (1992) Mathematical Simulation of Prothrombinase.  Methods Enzymol. 215:316-328. ( 1/1992 ) Link...
  Non-Scholarly Journals
  • D. S. Boskovic, T. Troxler and S. Krishnaswamy. (2005) Using the Fluorescence Resonance Energy Transfer Approach to Demonstrate Active Site-Independent Recognition of Substrates and Product by Bovine Prothrombinase.  The RLBL Newsletter 30:8-10. ( 3/2005 ) Link...