Citations

Infection increases propensity for thrombotic events. Gram negative pathogens release lipopolysaccharides (LPS), which initiate intracellular signaling cascades through activation of toll‐like‐receptor 4 (TLR4). Platelets contribute to defense from infection and to thrombosis, but little is known about how LPS affects platelet production, life span and reactivity. Platelet life span was determined by in vivo biotinylation in wild type (C57BL/10SnJ; WT) and TLR4 deficient (C57BL/10ScN; dTLR4) mice before and after a single intravenous injection of LPS (1ng – 500ng/25g body weight). Prior to LPS injection, the number of platelets and percentage of reticulated platelets in dTLR4 mice was less than in WT mice but there were no differences in platelet life span, indicating that loss of TLR4 affects platelet production and not life span. A single injection of LPS dose dependently increased percentages of P‐selectin positive platelets and of reticulated platelets but decreased the number of biotin positive platelets within 24hrs in WT but not in dTLR4 mice. Results suggest that LPS–simulated infection reduces life span of circulating platelet and increases production of more reactive (reticulated) platelets from megakaryocytes through TLR4 mechanisms. Thus, a single exposure to LPS represents a “priming” mechanism which could increase risk for thrombosis with infection. (Funded by NIH‐HL78638 and Mayo Fnd.)

Abstract Abstract 750 Background: DKK1 is a negative regulator of the Wnt signaling pathway in bone that is overexpressed in a subset of newly-diagnosed MM patients with osteolytic lesions as well as in refractory and relapsed patients. Expression also correlates with the number of osteolytic lesions in untreated MM patients. BHQ880 is a novel anti-DKK1 human monoclonal antibody. Alleviation of DKK1 inhibition by BHQ880 results in activation of the Wnt signaling pathway, leading to increased bone mass mediated via upregulation of osteoblasts in mice and monkeys. In murine models of MM bone disease; this anabolic activity of BHQ880 increased trabecular and cortical bone density. Lytic bone disease in MM is caused by osteoclast activation and osteoblast inhibition. Current approved therapies for the treatment of MM bone disease are focused on osteoclast inhibition (e.g., bisphosphonates) and BHQ880 therapy may be able to reverse the effects of DKK1-induced osteoblast inhibition. Therefore, dual therapy with zoledronic acid to decrease bone resorption and BHQ880 to increase new bone formation may provide an effective treatment strategy for MM bone disease. Methods: In the phase I portion of this phase I/II study, patients with relapsed or refractory MM with prior skeletal-related event (SRE) were treated with BHQ880 as an IV infusion Q28 days. Patients also received Zol (4 mg) and approved MM Tx (bortezomib not allowed). Bone markers and total DKK1 levels along with bone mineral density are being measured. Full PK profiles were collected during the first and second cycle, after which predose (trough) samples were collected to assess accumulation. Results: Ten pts (6:M, 4:F), median age: 66.5 yrs (range 41- 70), performance status-0(5 pts), 1(4 pts), 2 (1 pts) have been enrolled in the following dose levels (mg/kg) 3 (2 pts), 10 (starting dose level -4 pts), 20 (4 pts) and have been on treatment for 1 day to 5 28-day cycles. No BHQ880-related AE's have been observed to date. Bone mineral density (BMD) data from the first two patients treated at 10 mg show the following: a.) Pt 1 (hip +5.8%, spine N/A due to surgery, wrist -1.8%); b.) Pt 2 (hip -0.2%, spine +6.1%, wrist not done). The biomarker data from these patients show: a.) Pt 1 shows a maximal +98% change over baseline in PINP at day 15 post-treatment, while osteocalcin (OC) changes +17% at day 15, reaching a +56% change over baseline at cycle 4 day 1; uNTx/Cr changes – 43 % at cycle 2 day 15. b) Partial data from Pt 2 suggests a -44 % change in PINP and –73 % change in OC and a +73% change in uNTx/Cr. Baseline total DKK1 levels on 3 patients ranged from 8.8 to 21.5 ng/mL. Preliminary PK analysis is available in 1 out of 3 patients treated at 10mg/kg. The Cmax achieved after the first infusion was 165 ug/mL and 201 ug/mL after the second infusion. Calculated t1/2 was 188 hours after the first infusion and 254 hours after the second infusion. Regarding overall exposure, AUC0-672 hours after first infusion was 36081 hr*ng/mL and 48533 hr*ng/mL after the second infusion. Conclusion: BHQ880 given IV Q28 days appears to be well tolerated in combination with Zol and chosen MM Tx. Once safety of 40 mg/kg BHQ880 or the MTD dose has been determined, pts will be enrolled in the phase II portion to assess activity on a SRE endpoint. Updated safety, efficacy, bone density and biomarker data will be presented on all patients at the upcoming ASH meeting. Disclosures: Padmanabhan: Genentech: Consultancy, Honoraria; GSK: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. Off Label Use: BHQ880, a novel osteoblast activating, anti-DKK1 antibody. Dzik-Jurasz:Novartis: Employment, Equity Ownership. Gangolli:Novartis: Employment, Equity Ownership. Ettenberg:Novartis: Employment, Equity Ownership. Miner:Novartis: Employment, Equity Ownership. Bilic:Novartis: Employment, Equity Ownership. Whyte:Novartis: Employment, Equity Ownership. Mehdi:Novartis: Employment, Equity Ownership. Chiang:Novartis: Employment, Equity Ownership. Rae:Novartis: Employment, Equity Ownership. Shah:Novartis: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Millenium: Research Funding, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Elan: Consultancy. Giles:Novartis: Consultancy, Research Funding; BMS: Research Funding; Merck: Research Funding; Clavis: Research Funding. Stewart:Novartis: Consultancy; Amgen: Consultancy; Millenium: Consultancy, Research Funding; Proteolix: Consultancy; Celgene: Honoraria.

Thrombin Quick (TQ) may be isolated following treatment of Prothrombin Quick [Owen, et al, Mayo Clinic Proceedings, 53: 29-33, (1978)] with Taipan venom, phospholipid and ca2+. The clotting activity of TQ with fibrinogen is 1/200 that of nornar thrombin (T). The activation of Factors V and VIII, and the aggregation of platelets by TQ occurs with an effectiveness of about 1/50 that of thrombin. when incubated with antithrombin III, both T ad TQ fom inhibitor complexes as determined by dodecylsulfate gel electropheresis. Titration of T and TQ with the fluorescent inhibitor dansylarginine-4-ethylpiperidine amide indicates an equivalent number of active sites based on protein absorption at 280 nm. However, the two enzymes may be distinquished by the decreased fluorescence enhancement observed with TQ relative to T, indicating an increased polarity in the inhibitor binding site of TQ. With the substrate benzoylarginine ethylester, TQ has a Km = 4.5 × 10-5M and kcat= 6.93 compared to Km = 4.0 × 10-5M and kcat= 17.7 for T. This indicates that the defect in TQ esterase activity is in the catalytic mechanism itself and not in substrate binding. The rate of inhibition of TQ by diisopropylphosphofluoridate is decreased. Decreased acylation and deacylation rates for TQ relative to T are observed for hydrolysis of the active site titrant 4-methykl-umbelliferyl-p-guanidinobenzoate

Association of heparin non-covalently with bovine factor Xa was analyzed by Superose-12 gel chromatography. In 0.05 M NaCl, 0.02 M Tris, pH 7.5, DEGR-Xa (factor Xa inactivated by dans-Glu-Gly-Arg-CH2Cl) was eluted as a single, sharp peak at Ve/Vt=0-65 (elution volume/internal volume). Mixtures of heparin and DEGR-Xa were eluted as two partially resolved peaks of protein at Ve/Vt=0.59 and 0.65. The fraction of DEGFUXa in the leading peak was directly proportional to [heparin], and at 100 yM heparin the leading peak contained more than half the total protein. When 0.02 M HEPES was substituted for Tris a single, slightly broadened peak at Ve/Vt=0.64 was obtained on chromatography of 100 μM heparin and 10 μM DEGR-Xa. In a buffer system comprising 0.02 M Tris, 0.02 M HEPES, 0.03 M NaCl, pH 7.5, two peaks were eluted at Ve/Vt=0.59 and 0.65. Therefore, Tris increases the affinity of DEGR-Xa for heparin.Solutions buffered with Tris or HEPES were compared for effects on the kinetics of inhibition of factor Xa by antithrombin III-heparin. Reaction mixtures containing 1 nM factor Xa, 30 nM heparin and 600 nM antithrombin III were assayed with S-2222 at intervals of 2-10 sec. Reagent concentrations were chosen (a) to assure pseudo-first-order kinetics, (b) to have [heparin]<< Kq for factor Xa-heparin, and (c) to bind virtually all available heparin to antithrombin III. The same second-order rate constant, Kobs=2.5×107 M−1s−1, was obtained in both buffer systems. We conclude that the association of factor Xa with heparin observed directly by gel chromatography does not contribute to the reaction rate of factor Xa with antithrombin III-heparin.

SummaryReversibility in vivo of acute platelet thrombosis in response to specific anticoagulants is analyzed with thrombi that develop in segments (1 cm) of porcine carotid arteries externally crushed with a hemostat. Most thrombi fill the lumens of the injured segments (ca. 1 cm × 3 mm, 1 × w) within 30 min and comprise masses of platelets interpenetrated with neutrophil-lined seepage channels of blood. Continuous quantitative assay of thrombus mass is provided by a gamma detector placed over the injured segments to collect counts from 111In-labeled platelets. Thrombi established 30 min after injury, otherwise stable for 6 h, clear during 30-60 min of continuous infusion of either hirudin, tick anticoagulant or activated porcine protein C, or intermittent activation of endogenous protein C with a latent thrombin reagent. Anticoagulant dose-dependence of thrombus clearance is established for hirudin between 0.01 and 1.0 mg/kg/min. Thrombi become progressively refractory to hirudin between 0.5 and 6 h after injury. Neither heparin nor low-molecular-weight heparin in full (clinical) anticoagulant doses yield significant dethrombosis. It is concluded that, within time limits, controlled thrombin generation in platelet thrombi maintains platelet cohesion without catalyzing irreversible platelet aggregation or clotting of fibrinogen.

SummaryThe relative contribution of plasmin, miniplasmin, PMN-elastase and cathepsin G to the fibrin-gel dissolution is studied. The global kcat/KM ratios are determined as a measure of the fibrinolytic catalytic efficiency using spectrophotometric kinetic analysis of the competition between fibrin and synthetic peptide substrates for the proteases, turbi-dimetric assay for fibrin dissolution and gel-filtration of the partially degraded fibrin. When the substrate is fibrin polymerized in the presence of 3 mM Ca2+, the value of this ratio is 4.3 × 105 M-1·s-1 for plasmin, 1.9 × 105 M-1·s-1 for miniplasmin, 5.0 × 104 M-1·s-1 for PMN-elastase and 2.2 × 103 M-1·s-1 for cathepsin G. When fibrin is polymerized without addition of Ca2+, the kcat/KM values are increased by a factor of 2.3 for plasmin, 2.0 for miniplasmin and 1.6 for cathepsin G, whereas that of PMN-elastase is unchanged. Progressive crosslinking of fibrin decreases the catalytic action of all studied proteases, but no change in their relative contribution to fibrinolysis is observed. When plasmin inhibitor (at physiological concentration) is also crosslinked to fibrin, the most efficient fibrinolytic enzymes are miniplasmin and PMN-elastase. The effect of 6-aminohexanoate on the formation of fibrin degradation products by plasmin and miniplasmin suggests that the high-affinity lysine binding site in the N-terminal kringle domain of plasmin is involved in the interactions with the native polymerized fibrin, whereas the fifth kringle found in both enzymes participates in binding to newly exposed lysine residues. These results provide a quantitative basis for the evaluation of fibrinolytic efficiency and support the concept of synergistic fibrinolysis.

SummaryThe plasmin substrate, H-D-norleucyl-hexahydrotyrosyl lysine-p-nitroanilide (Spectrozyme-PL), was found to be equiva lent to 6-aminohexanoate as an enhancer of porcine and human plasminogen activation by urokinase and of removal of the 1-77 peptide of plasminogen by plasmin. Activation of plasminogen lacking kringles 1-4, on the other hand, was not influenced by Spectrozyme PL. Although the rate of activation of human plasminogen and the modification of human plasminogen by plasmin are faster by an order of magnitude than that of the activation and modification of porcine plasminogen, both reactions in the human zymogen, the hydrolysis at arg561-val562 and at lys77lys78, are accelerated by Spectrozyme PL. The findings indicate that kinetic interpretation of plasminogen activation in solutions containing substrates, where the substrate has been incorporated to inhibit feedback proteolysis by plasmin, must account for the cofactor activity as well as the inhibitory activity of the substrate.

Differences in the aggregation and release of growth factors including matrix metalloproteinases (MMPs) after loss of ovarian hormones could contribute to an exaggerated response to injury in arteries of ovariectomized animals. Therefore, experiments were designed to compare aggregation, dense granular ATP release, expression of MMPs (MMP-2, MMP-9, and MMP-14) and tissue inhibitors of metalloproteinase (TIMP-1 and TIMP-2) in circulating platelets from sexually mature (7 mo old) gonadally intact and ovariectomized (4 wk) female pigs. Numbers of circulating platelets did not change after ovariectomy, but the percentage of reticulated platelets increased significantly. Platelet aggregation and dense granular ATP secretion also increased significantly with ovariectomy. In platelet lysates, active MMP-2 increased, whereas MMP-14 significantly decreased, after ovariectomy; the expression of TIMP-1, TIMP-2, and P-selectin did not change. These results suggest that platelet turnover, aggregation, and ATP secretion increase with ovariectomy. Also, ovarian hormones selectively regulate the expression and activity of MMPs in porcine platelets. Increased platelet aggregation and activity of MMP-2 would alter platelet-platelet and platelet-vessel wall interactions, contributing to an exaggerated response to injury with loss of ovarian hormones.