Effects of the pan-selectin antagonist bimosiamose (TBC1269) in experimental human endotoxemia.

Selectins mediate the adhesion of leukocytes to activated endothelial cells and activated platelets. In addition to these cell-to-cell interactions, they influence the fibrin content and size of venous thrombi in different animal models. However, the exact role of selectins in human endotoxemia still remains unclear. We aimed to investigate the effect of selectin inhibition in lipopolysaccharide (LPS)-induced tissue factor (TF)-dependent activation of coagulation in a well-standardized model of human endotoxemia. To explore whether selectin blockade attenuates LPS-induced coagulation in humans, we performed a randomized, double-bind placebo-controlled crossover trial in 16 healthy male volunteers. All subjects received 2 ng/kg of LPS and, 10 min thereafter, a 15-min infusion of either 30 mg/kg of the pan-selectin antagonist bimosiamose or equal volumes of placebo in random order, with a washout period of 6 weeks between both periods. Treatment with bimosiamose had no significant effect on LPS-induced TF expression, as quantified by TF mRNA levels, or on LPS-induced coagulation response, reflected by increases in plasma thrombin-antithrombin (TAT) complexes and prothrombin fragment (F1 + 2) levels. Furthermore, bimosiamose did not affect the LPS-dependent changes in leukocyte subpopulations or the increase in platelet-leukocyte aggregates, as determined in the level of CD41+ monocytes. Finally, neither the LPS-induced release of tumor necrosis factor, interleukin 6, leukocyte expression of CD11b, nor intercellular adhesion molecule 1 were affected by administration of bimosiamose. The pan-selectin antagonist bimosiamose does not attenuate TF-triggered coagulation or inflammation in human endotoxemia. This indicates a minor influence of this selectin antagonist in this model. In addition, infusion of bimosiamose was safe and well tolerated in human endotoxemia.

Recombinant human antithrombin inhibits thrombin formation and interleukin 6 release in human endotoxemia.

We hypothesized that infusion of recombinant human antithrombin without concomitant heparin would have dose-dependent anticoagulant properties and potentially decrease endotoxin (lipopolysaccharide [LPS])-induced cytokine production. This was a randomized, double-blind, placebo-controlled study in parallel groups enrolling 30 healthy male volunteers. The active treatment groups received infusions of recombinant human antithrombin to increase antithrombin levels to 200% and 500% before infusion of 2 ng/kg endotoxin (LPS). Infusion of antithrombin dose-dependently decreased coagulation (P < .01 by repeated-measures ANOVA): peak levels of prothrombin fragment (1.8 nmol/L [95% confidence interval (CI), 1.3-2.3 nmol/L] in the 500% antithrombin group and 4.4 nmol/L [95% CI, 2.7-6.2 nmol/L] in the placebo group at 4 hours), thrombin antithrombin complexes (12 microg/L [95% CI, 8-16 microg/L] in the 500% antithrombin group and 34 microg/L [95% CI, 20-48 microg/L] in the placebo group at 4 hours), and D-dimer (0.2 microg/L [95% CI, 0.1-0.2 microg/L] in the 500% antithrombin group and 0.5 microg/L [95% CI, 0.4-0.7 microg/L] in the placebo group). Recombinant human antithrombin decreased peak interleukin-6 levels by 40% (222 pg/mL [95% CI, 148-295 pg/mL] and 216 pg/mL [95% CI, 112-320 pg/mL] in the 500% and 200% antithrombin groups, respectively, versus 357 pg/mL [95% CI, 241-474 pg/mL] in the placebo group; P < .001 by ANOVA). Finally, infusion of recombinant human antithrombin rapidly and transiently decreased neutrophil counts (by 19% [95% CI, 8%-30%] in the 500% antithrombin group versus 6% [95% CI, 1%-10%] in the placebo group, P = .002 by Kruskal-Wallis ANOVA) and monocyte counts (by 30% [95% CI, 16%-44%] in the 500% antithrombin group and 18% [95% CI, 9%-28%] in the 200% antithrombin group versus 8% [95% CI, 5%-20%] in the placebo group, P = .04) before LPS challenge, indicating that recombinant human antithrombin directly interacts with these leukocyte subsets. In summary, recombinant human antithrombin dose-dependently inhibited tissue factor-triggered coagulation. Effects on leukocytes and inhibition of interleukin-6 release seem to represent specific pharmacodynamic properties of recombinant human antithrombin.

Pharmacokinetics (PK) of S/D treated anti-D immunoglobulin after intramuscular injection in healthy volunteers: gender differences in PK.

The aim of this study was to investigate the pharmacokinetic profile of the new solvent/detergent (S/D) formulation of an anti-D IgG preparation, and to evaluate gender differences. RhD-negative subjects (m/f=10/8) received a single i.m. injection of 250 microg anti-D (Partobulin SDF). There was a rapid increase in median anti-D titers over the first 2 days, followed by a plateau from days 2-7. Interestingly, women had a higher maximum concentration (Cmax) of anti-D and a lower volume of distribution at steady state (Vss) than men. The half-life calculated in this study was 23 days. Thus, results are comparable to published data of the non-S/D treated predecessor product. Because of the observed gender differences in the pharmacokinetics we recommend to pursue the evaluation of sex differences in the pharmacokinetics of other antibodies during early phase drug development.

Additive effects between platelet concentrates and desmopressin in antagonizing the platelet glycoprotein IIb/IIIa inhibitor eptifibatide.

BACKGROUND: Platelet (PLT) glycoprotein (GP) IIb/IIIa receptor antagonists have demonstrated efficacy in decreasing ischemic complications of percutaneous coronary intervention and/or unstable angina. In case of bleeding, the drug can be stopped and PLT transfusions can be given. STUDY DESIGN AND METHODS: This crossover study tested the additive effects of PLT concentrates (PCs) after desmopressin (DDAVP) infusion in antagonizing the anti-PLT effects of GPIIb/IIIa inhibitors and aspirin. After eptifibatide and aspirin infusion (at standard dosages), 10 healthy volunteers received DDAVP or placebo. Thereafter, increasing amounts of PLTs from fresh single-donor apheresis concentrates were added in vitro to blood samples of all volunteers to increase PLT counts by 30 x 10(9), 60 x 10(9), or 120 x 10(9) per L. RESULTS: Adding platelets in vitro further improved PLT function after DDAVP: it shortened collagen-adenosine diphosphate closure times (p < 0.01), to normal ranges as measured by the PLT function analyzer (PFA-100). In contrast, normal PLT function could not be restored even when PLT counts were increased by 50 percent (120 x 10(9)/L) in the placebo group. CONCLUSION: Combined use of PLTs from fresh apheresis PC and DDAVP additively enhances recovery of normal PLT function after eptifibatide infusion. Such a strategy may help to avoid excessive transfusion of PC.

Changes in ADAMTS13 (von-Willebrand-factor-cleaving protease) activity after induced release of von Willebrand factor during acute systemic inflammation.

Von Willebrand factor (VWF) is synthesized in endothelial cells, stored in the form of high molecular weight multimers and released after stimulation. After release, the multimers are cleaved by ADAMTS13 (von-Willebrand-factor-cleaving protease). We studied healthy volunteers in a double-blind, placebo controlled inflammation model. Ten male volunteers received 2 ng/kg endotoxin intravenously, and 5 volunteers placebo. Endotoxin infusion induced systemic inflammation and coagulation activation. After 4 hours the observed increase in neutrophils reached a maximum (273+/-34% of baseline; mean+/-SEM) and the platelet count dropped (81+/-2%). These parameters returned to baseline values after 24 hours. VWF antigen increased to 259+/-16% of baseline after 4 hours, remained elevated (192+/-15%) after 24 hours and returned to baseline after 7 days. Unusually large VWF multimers occurred in the plasma 4 hours after endotoxin infusion. ADAMTS13 activity (measured with a collagen-binding assay) decreased to 64+/-5% of baseline (P<0.001) after 4 hours, was still reduced after 24 hours (86+/- %; P=0.008) and returned to normal after 7 days. VWF multimer analysis showed pronounced satellite bands in the 4-hour samples, indicating cleavage of VWF by ADAMTS13. No apparent changes of the analyzed parameters were observed in the placebo group. The reciprocal course of ADAMTS13 and VWF after short-term VWF release induced by systemic inflammation is similar to that observed after induction of VWF release by desmopressin.

Angiotensin receptor blockade decreases markers of vascular inflammation.

A protective role against atherosclerosis can be attributed to angiotensin converting enzyme inhibitors (ACE-I), since they have been shown to reduce mortality in patients at cardiovascular risk. Since plasma levels of adhesion molecules are considered surrogate markers of endothelial cell activation and atherogenesis, we compared the levels of adhesion molecules after treatment with the ACE-I enalapril or the direct angiotensin- receptor antagonist losartan or placebo. In a randomized, controlled trial, 21 hypercholesterolemic volunteers received 50 mg/d losartan or 20 mg/d enalapril or placebo for twelve weeks. Plasma levels of circulating intercellular adhesion molecule-1 (cICAM-1), vascular adhesion molecule-1 (cVCAM-1), and E-selectin (cE-SEL) were measured by ELISA. Surface expression of ICAM-1 on circulating leukocytes was determined by flow cytometry. Enalapril and losartan but not placebo induced a small but stable decrease of cICAM-1 and cVCAM-1, while cE-SEL and leukocyte expression of ICAM-1 remained unchanged. The lowering of plasma adhesion molecules may indicate an antiatherogenic effect of angiotensin II blockade in hypercholesterolemia. While such preventive effect will have to be proven in clinical trials, our results do not support a preference for either enalapril or losartan with regard to their possible vasoprotective role.

Defects in the prostaglandin system--what is known?

The central role of prostaglandins as local mediators is well accepted. Molecular biology and in particular knock-out mice models teach us a lot on mechanisms and eventual biological consequences. Despite the broad basic knowledge available, human data on defects in the prostaglandin system are extremely rare. Why? Don't we search for them? Are they of clinical relevance? What is their prevalence, the outcome? How to treat, if possible? For this purpose we are planning a platform and databank to improve knowledge, pool information and allow exchange of probes. All interested people are invited to join.

Regulation of protease-activated receptor 1 (PAR1) on platelets and responsiveness to thrombin receptor activating peptide (TRAP) during systemic inflammation in humans.

Thrombin is a coagulation protease that activates platelets, endothelial cells, leukocytes and mesenchymal cells. Thrombin signaling is mediated at least in part by protease-activated receptors (PARs). As little is known about the in vivo regulation of PAR1, this study aimed to characterize the effects of systemic thrombin formation during human endotoxemia on the regulation of PAR1 and the associated responsiveness of human platelets to thrombin receptor activating peptide (TRAP). Endotoxin (2 ng/kg) was infused into 40 healthy men to study the regulation of PAR1 in systemic human inflammation. The SPAN12 antibody was used to determine the in vivo regulation of PAR1. To measure whether modulation of the PAR1 receptor may be associated with altered platelet reactivity, whole blood was stimulated with TRAP ex vivo. Thrombin generation was determined by prothrombin (F(1+2)) fragment. F(1+2) levels increased almost 9-fold from 0.5+/-0.1 nmol/L to 4.5+/-1.9 nmol/L at 4 h (p<0.001). PAR1 decreased by approximately 8% (p<0.001) within 2 h after endotoxin infusion and stayed at those levels until 6 h. Concomitantly, TRAP induced P-selectin expression maximally decreased by 18% (p<0.001) at 6 h. In conclusion, PAR1 expression is down-regulated on platelets during systemic thrombin formation induced by inflammation in humans which results in decreased responsiveness to subsequent stimulation of the PAR1 receptor.

Desmopressin antagonizes the in vitro platelet dysfunction induced by GPIIb/IIIa inhibitors and aspirin.

Whereas bleeding is the most frequent adverse event encountered in patients receiving glycoprotein (GP) IIb/IIIa inhibitors, there are currently no recommendations for how to treat such patients. The present study tested the hypothesis that infusion of desmopressin (DDAVP) reverses the in vitro platelet dysfunction induced by GPIIb/IIIa inhibitors (+l-aspirin). Study group 1 (10 healthy volunteers) received a DDAVP infusion to establish dose-response curves for the in vitro inhibition of platelet function by eptifibatide, abciximab, and tirofiban together with l-aspirin before and after DDAVP. In a randomized, double-blind, placebo-controlled, crossover study (group 2) volunteers received l-aspirin and a standard eptifibatide infusion. Thereafter, DDAVP or a physiologic saline infusion was given over 30 minutes. In group 1, all GPIIb/IIIa inhibitors prolonged collagen-epinephrine (CEPI) and collagen-adenosine diphosphate (CADP) closure times (CTs), measured with the platelet function analyzer 100 (PFA-100). DDAVP caused a shift in the concentration response curves to the right of all 3 GPIIb/IIIa inhibitors. In group 2, DDAVP accelerated the normalization of CADP-CT and CEPI-CT after the stop of eptifibatide infusion with a maximum effect at 1.5 hours to 2 hours. In contrast, CEPI-CT remained above normal in the placebo group for more than 4 hours. In conclusion, DDAVP accelerates normalization of the in vitro platelet dysfunction induced by GPIIb/IIIa inhibitors (+l-aspirin).

Recombinant human activated protein C (rhAPC; drotrecogin alfa [activated]) has minimal effect on markers of coagulation, fibrinolysis, and inflammation in acute human endotoxemia.

Inflammatory and procoagulant host responses are closely related in sepsis. The protein C pathway serves as a regulatory pathway with anti-inflammatory and anticoagulant properties. Recently, recombinant human activated protein C (rhAPC) was shown to reduce mortality in severe sepsis. Nevertheless, the effects of rhAPC in humans are still ill defined. The infusion of low endotoxin doses into humans provides a standardized model to study inflammatory and hemostatic mechanisms. Thus, we investigated whether rhAPC acts as an anticoagulant or anti-inflammatory drug in human endotoxemia. There were 24 volunteers randomized to receive either 24 microg/kg per hour rhAPC or placebo intravenously for 8 hours. Lipopolysaccharide (LPS, 2 ng/kg) was administered 2 hours after starting the infusions. rhAPC decreased basal tissue factor (TF)-mRNA expression, and thrombin formation and action. In contrast, rhAPC did not significantly blunt LPS-induced thrombin generation. Consistently, rhAPC did not reduce LPS-induced levels of TF-mRNA or D-dimer and had no effect on fibrinolytic activity or inflammation. Finally, endogenous APC formation was enhanced during endotoxemia and appeared to be associated with inflammation rather than thrombin formation. In conclusion, even low-grade endotoxemia induces significant protein C activation. Infusion of rhAPC decreases "spontaneous" activation of coagulation but does not blunt LPS-induced, TF-mediated coagulation in healthy volunteers, which is in contrast to a number of anticoagulants.

Changes in von Willebrand factor-cleaving protease (ADAMTS13) activity after infusion of desmopressin.

von Willebrand factor-cleaving protease (ADAMTS13) cleaves von Willebrand factor (VWF) and regulates its physiologic function. To investigate the relation between ADAMTS13 activity and VWF, we compared ADAMTS13 activity with the VWF-related parameters VWF antigen (VWF:Ag), VWF collagen-binding activity (VWF:CBA), VWF-propeptide, proVWF, and VWF multimeric composition in 10 healthy volunteers and 3 patients with type 1 von Willebrand disease before and after infusing 0.3 microg/kg desmopressin. The VWF-related parameters in the volunteers increased 60 minutes after start of infusion by 3.7-fold for VWF:Ag, 7.2-fold for propeptide, and 2.2-fold for VWF:CBA. Unusually large VWF multimers and traces of proVWF appeared. The ADAMTS13 activity decreased to about half the initial value. After 24 hours values returned to baseline. Patients with type 1 von Willebrand disease showed similar results. We conclude that the inverse correlation between ADAMTS13 and VWF-related parameters suggests a consumption of ADAMTS13 after the desmopressin-induced release of higher multimers of VWF.

Point of care measurement of lepirudin and heparin anticoagulation during systemic inflammation.

BACKGROUND: The number of indications for recombinant human hirudin lepirudin therapy has increased in recent years, and now includes acute coronary syndromes and heparin-induced thrombocytopenia. Hence, point of care monitoring appears desirable for therapy with lepirudin. As CoaguChek Plus (CCP) provides a rapid bedside test to monitor therapy with other anticoagulants, we aimed to determine its suitability for lepirudin therapy. METHODS: Forty-four healthy volunteers received a 2 ng/kg endotoxin infusion (to induce coagulation) together with clinically relevant doses of lepirudin or heparin in a prospective, placebo-controlled, randomised fashion. Measurements of CCP-partial thromboplastin time (aPTT) were compared to laboratory STA-aPTT. RESULTS: As expected, baseline values of CCP-aPTT were shorter than STA-aPTT. Lepirudin increased CCP-aPTT 3-fold, and STA-aPTT 2-fold 1 h after bolus infusion. During lepirudin infusion, the correlation between CCP-aPTT and STA-aPTT was excellent (r=0.86-0.92). Both methods were equally sensitive to over-anticoagulation with heparin. Acute systemic inflammation had little effects on CCP-aPTT. CONCLUSION: CCP-aPTT is suitable for longitudinal point of care monitoring of lepirudin therapy. As baseline values of CCP-aPTT are shorter than STA-aPTT, it is recommended not to indiscriminately change between methods in the follow-up of individual patients.