Antifibrinolytic Efficacy of Truncated L17R Mutant of Kunitz Domain 1 (KD1) of Tissue Factor Pathway Inhibitor-2 (TFPI-2): Comparison with Lysine Analogues

Abstract 855 Previously, we demonstrated that changing residue Leu17 (BPTI/Aprotinin numbering) to Arg in Kunitz domain 1 (73-residue KD1-L17R) of TFPI-2 abolishes its anticoagulant functions and enhances its plasmin inhibition (Bajaj et al., J Biol Chem 286, 4329–4340, 2011). In that study we used the entire KD1 domain, which in addition to the core structural homologous region of BPTI (58 residues) included 9 residues on the N-terminal and 6 residues on the C-terminal side of the protein. Conformation of these 15 residues may be different in the isolated KD1-domain as compared to the complete TFPI-2 molecule. Thus, these residues could be potentially immunogenic. To address these concerns, we investigated weather N- and C-terminal regions of 73-residue KD1-L17R could be cleaved upon prolonged incubation with thrombin (IIa). Incubation of 73-residue KD1-L17R with IIa for 72 hrs yielded smaller version(s) of KD1-L17R as analyzed by SDS-PAGE. N-terminal sequence and MALDI-TOF/ESI mass spectrometry analyses revealed three closely related species present in the truncated KD1-L17R preparations (Fig. 1). Species 1 has Gly-Asn-Asn as the amino terminus and Val-Pro-Lys as the C-terminus. Species 2 and 3 are similar to species 1 except species 2 is produced after losing Gly and Asn from the N-terminus, whereas species 3 is produced after losing Val-Pro-Lys from the C-terminus. Thus, all three species have the intact core Kunitz domain with minor variations at the N- and C-terminus regions. Further, these species are cleaved at the viable albeit very slow IIa-cleavage sites; herein, these species are collectively referred to as truncated KD1-L17R. A plausible mechanism for proteolysis at these cleavage sites is shown in Fig. 2. Similar to the 73-residue KD1-L17R, the truncated preparations did not inhibit (Ki > 3 μM) plasma kallikrein, factor (F) XIa, FVIIa/soluble tissue factor, FXa, activated protein C, tissue plasminogen activator (tPA), IIa and IIa/soluble thrombomodulin. Importantly, the truncated KD1-L17R preparations inhibited plasmin with Ki ∼1.2 nM. Further, the truncated KD1-L17R inhibited tPA-induced plasma clot fibrinolysis with an apparent IC50 of ∼0.37 μM, a value similar to that obtained with the 73-residue KD1-L17R and BPTI. Two lysine analogues, Epsilon amino caproic acid (EACA) and tranexamic acid (TE) inhibited tPA-induced plasma clot fibrinolysis with an apparent IC50 of ∼80 μM and ∼20 μM, respectively. Further, efficacy of truncated KD1-L17R was tested in a mouse liver laceration model of bleeding. As compared to saline, the amount of blood loss was reduced by ∼65% by truncated KD1-L17R (N=6, p 0.001), ∼70% by BPTI (N=10, p 0.003), ∼52% by TE (N=10, p 0.019) and ∼25% by EACA (N=16, p 0.03). We also observed seizures in four (25%) of the animals treated with a single dose of EACA. In conclusion, truncated KD1-L17R is an effective antifibrinolytic agent similar to the 73-residue KD1-L17R and BPTI/Aprotinin. Although lysine analogues are relatively effective in reducing blood loss, EACA caused seizures in our studies. These observations are consistent with recent reports that one of the major side effects of lysine analogues is seizures (Martin et al., J Cardiothorac Vasc Anesth 25, 20–25, 2011; Koster and Schirmer, Curr Opin Anaesthesiol 24, 92–97, 2011). We conclude that truncated KD1-L17R may serve as an excellent alternative to BPTI and lysine analogues in preventing blood loss during major surgeries including coronary artery bypass graft (CABG) surgery. We are currently expressing the 60-residue KD1-L17R (NH2Asn-Ala-Glu······Ile-Glu-Lys) protein for further efficacy studies. We are also generating additional mutant(s) on the 60-residue KD1-L17R molecule for achieving increased plasmin potency without provoking anticoagulant functions. Supported By HL89661 and HL36365. Disclosures: No relevant conflicts of interest to declare.

Prothrombin activation on the activated platelet surface optimizes expression of procoagulant activity

Effective hemostasis relies on the timely formation of α-thrombin via prothrombinase, a Ca2+-dependent complex of factors Va and Xa assembled on the activated platelet surface, which cleaves prothrombin at Arg271 and Arg320. Whereas initial cleavage at Arg271 generates the inactive intermediate prethrombin-2, initial cleavage at Arg320 generates the enzymatically active intermediate meizothrombin. To determine which of these intermediates is formed when prothrombin is processed on the activated platelet surface, the cleavage of prothrombin, and prothrombin mutants lacking either one of the cleavage sites, was monitored on the surface of either thrombin- or collagen-activated platelets. Regardless of the agonist used, prothrombin was initially cleaved at Arg271 generating prethrombin-2, with α-thrombin formation quickly after via cleavage at Arg320. The pathway used was independent of the source of factor Va (plasma- or platelet-derived) and was unaffected by soluble components of the platelet releasate. When both cleavage sites are presented within the same substrate molecule, Arg271 effectively competes against Arg320 (with an apparent IC50 = 0.3μM), such that more than 90% to 95% of the initial cleavage occurs at Arg271. We hypothesize that use of the prethrombin-2 pathway serves to optimize the procoagulant activity expressed by activated platelets, by limiting the anticoagulant functions of the alternate intermediate, meizothrombin.

The Interaction of Na+ and K+ in Voltage-gated Potassium Channels

Voltage-gated potassium (K+) channels are multi-ion pores. Recent studies suggest that, similar to calcium channels, competition between ionic species for intrapore binding sites may contribute to ionic selectivity in at least some K+ channels. Molecular studies suggest that a putative constricted region of the pore, which is presumably the site of selectivity, may be as short as one ionic diameter in length. Taken together, these results suggest that selectivity may occur at just a single binding site in the pore. We are studying a chimeric K+ channel that is highly selective for K+ over Na+ in physiological solutions, but conducts Na+ in the absence of K+. Na+ and K+ currents both display slow (C-type) inactivation, but had markedly different inactivation and deactivation kinetics; Na+ currents inactivated more rapidly and deactivated more slowly than K+ currents. Currents carried by 160 mM Na+ were inhibited by external K+ with an apparent IC50 <30 μM. K+ also altered both inactivation and deactivation kinetics of Na+ currents at these low concentrations. In the complementary experiment, currents carried by 3 mM K+ were inhibited by external Na+, with an apparent IC50 of ∼100 mM. In contrast to the effects of low [K+] on Na+ current kinetics, Na+ did not affect K+ current kinetics, even at concentrations that inhibited K+ currents by 40–50%. These data suggest that Na+ block of K+ currents did not involve displacement of K+ from the high affinity site involved in gating kinetics. We present a model that describes the permeation pathway as a single high affinity, cation-selective binding site, flanked by low affinity, nonselective sites. This model quantitatively predicts the anomalous mole fraction behavior observed in two different K+ channels, differential K+ and Na+ conductance, and the concentration dependence of K+ block of Na+ currents and Na+ block of K+ currents. Based on our results, we hypothesize that the permeation pathway contains a single high affinity binding site, where selectivity and ionic modulation of gating occur.

Inhibitory action of somatostatin on isolated gastric glands and parietal cells

The action of somatostatin in vitro was characterized using glands and parietal cells isolated from rabbit gastric mucosa. In the presence of the reducing agent dithiothreitol, somatostatin was found to inhibit gastrin- and histamine-stimulated acid formation in glands as measured by [14C]aminopyrine (AP) accumulation and oxygen consumption, both measurements that appear to be reliable indexes of parietal cell acid formation. In glands the inhibition of the secretory response to gastrin was more potent (60-80%) than that to histamine (15-25%). The kinetics of somatostatin inhibition of responses to both agents were noncompetitive. The apparent IC50 for the partial somatostatin inhibition of histamine-stimulated AP accumulation was similar to that for gastrin (approx 3 X 10(-9) M) when maximum concentrations of histamine (10(-4) M) or gastrin (10(-7) M) were used. The inhibitory action of somatostatin appeared to be specific, inasmuch as this peptide had no significant effect on basal secretion or secretion stimulated by carbachol, dibutyryl cAMP, cholera toxin, or elevated extracellular K+. In purified parietal cell preparations, somatostatin inhibited histamine- but not gastrin-stimulated AP accumulation. Moreover, the inhibition of histamine-stimulated AP accumulation in parietal cells was more pronounced than in glands. These results suggest that somatostatin acts directly on parietal cells to inhibit histamine activation of H+ secretion. Somatostatin also acts indirectly to inhibit gastrin, perhaps by blocking the release of histamine from paracrine- or endocrinelike cells present in the glands.