Mercury free microscopy: an opportunity for core facility directors.

Mercury Free Microscopy (MFM) is a new movement that encourages microscope owners to choose modern mercury free light sources to replace more traditional mercury based arc lamps. Microscope performance is enhanced with new solid state technologies because they offer a more stable light intensity output and have a more uniform light output across the visible spectrum. Solid state sources not only eliminate mercury but also eliminate the cost of consumable bulbs (lifetime ∼200 hours), use less energy, reduce the instrument down time when bulbs fail and reduce the staff time required to replace and align bulbs. With lifetimes on the order of tens of thousands of hours, solid state replacements can pay for themselves over their lifetime with the omission of consumable, staff (no need to replace and align bulbs) and energy costs. Solid state sources are also sustainable and comply with institutional and government body mandates to reduce energy consumption, carbon footprints and hazardous waste. MFM can be used as a mechanism to access institutional financial resources for sustainable technology through a variety of stakeholders to defray the cost to microscope owners for the initial purchase of solid state sources or the replacement cost of mercury based sources. Core facility managers can take a lead in this area as "green" ambassadors for their institution by championing a local MFM program that will save their institution money and energy and eliminate mercury from the waste stream. Managers can leverage MFM to increase the visibility of their facility, their impact within the institution, and as a vital educational resource for scientific and administrative consultation.

Active site-labeled prothrombin inhibits prothrombinase in vitro and thrombosis in vivo.

Mouse and human prothrombin (ProT) active site specifically labeled with D-Phe-Pro-Arg-CH(2)Cl (FPR-ProT) inhibited tissue factor-initiated thrombin generation in platelet-rich and platelet-poor mouse and human plasmas. FPR-prethrombin 1 (Pre 1), fragment 1 (F1), fragment 1.2 (F1.2), and FPR-thrombin produced no significant inhibition, demonstrating the requirement for all three ProT domains. Kinetics of inhibition of ProT activation by the inactive ProT(S195A) mutant were compatible with competitive inhibition as an alternate nonproductive substrate, although FPR-ProT deviated from this mechanism, implicating a more complex process. FPR-ProT exhibited ∼10-fold more potent anticoagulant activity compared with ProT(S195A) as a result of conformational changes in the ProT catalytic domain that induce a more proteinase-like conformation upon FPR labeling. Unlike ProT and ProT(S195A), the pathway of FPR-ProT cleavage by prothrombinase was redirected from meizothrombin toward formation of the FPR-prethrombin 2 (Pre 2)·F1.2 inhibitory intermediate. Localization of ProT labeled with Alexa Fluor® 660 tethered through FPR-CH(2)Cl ([AF660]FPR-ProT) during laser-induced thrombus formation in vivo in murine arterioles was examined in real time wide-field and confocal fluorescence microscopy. [AF660]FPR-ProT bound rapidly to the vessel wall at the site of injury, preceding platelet accumulation, and subsequently to the thrombus proximal, but not distal, to the vessel wall. [AF660]FPR-ProT inhibited thrombus growth, whereas [AF660]FPR-Pre 1, lacking the F1 membrane-binding domain did not bind or inhibit. Labeled F1.2 localized similarly to [AF660]FPR-ProT, indicating binding to phosphatidylserine-rich membranes, but did not inhibit thrombosis. The studies provide new insight into the mechanism of ProT activation in vivo and in vitro, and the properties of a unique exosite-directed prothrombinase inhibitor.

A catalytic domain exosite (Cys527-Cys542) in factor XIa mediates binding to a site on activated platelets.

The zymogen, factor XI, and the enzyme, factor XIa, interact specifically with functional receptors on the surface of activated platelets. These studies were initiated to identify the molecular subdomain within factor XIa that binds to activated platelets. Both factor XIa (Ki approximately 1.4 nM) and a chimeric factor XIa containing the Apple 3 domain of prekallikrein (Ki approximately 2.7 nM) competed with [125I]factor XIa for binding sites on activated platelets, suggesting that the factor XIa binding site for platelets is not located in the Apple 3 domain which mediates factor XI binding to platelets. The recombinant catalytic domain (Ile370-Val607) inhibited the binding of [125I]factor XIa to the platelets (Ki approximately 3.5 nM), whereas the recombinant factor XI heavy chain did not, demonstrating that the platelet binding site is located in the light chain of factor XIa. A conformationally constrained cyclic peptide (Cys527-Cys542) containing a high-affinity (KD approximately 86 nM) heparin-binding site within the catalytic domain of factor XIa also displaced [125I]factor XIa from the surface of activated platelets (Ki approximately 5.8 nM), whereas a scrambled peptide of identical composition was without effect, suggesting that the binding site in factor XIa that interacts with the platelet surface resides in the catalytic domain near the heparin binding site of factor XIa. These data support the conclusion that a conformational transition accompanies conversion of factor XI to factor XIa that conceals the Apple 3 domain factor XI (zymogen) platelet binding site and exposes the factor XIa (enzyme) platelet binding site within the catalytic domain possibly comprising residues Cys527-Cys542.

Factor XI, but not prekallikrein, blocks high molecular weight kininogen binding to human umbilical vein endothelial cells.

Previous studies on the interaction of high molecular weight kininogen (HK) with endothelial cells have reported a large number of binding sites (106-107 sites/cell) with differing relative affinities (KD = 7-130 nm) and have implicated various receptors or receptor complexes. In this study, we examined the binding of HK to human umbilical vein endothelial cells (HUVEC) with a novel assay system utilizing HUVEC immobilized on microcarrier beads, which eliminates the detection of the high affinity binding sites found nonspecifically in conventional microtiter well assays. We report that HK binds to 8.5 x 104 high affinity (KD = 21 nm) sites per HUVEC, i.e. 10-100-fold fewer than previously reported. Although HK binding is unaffected by the presence of a physiological concentration of prekallikrein, factor XI abrogates HK binding to HUVEC in a concentration-dependent manner. Disruption of the naturally occurring complex between factor XI and HK by the addition of a 31-amino acid peptide mimicking the factor XI-binding site on HK restored HK binding to HUVEC. Furthermore, HK inhibited thrombin-stimulated von Willebrand factor release by HUVEC but not thrombin receptor activation peptide (SFLLRN-amide)-stimulated von Willebrand factor release. Factor XI restored the ability of thrombin to stimulate von Willebrand factor release in the presence of low HK concentrations. These results suggest that free HK, or HK in complex with prekallikrein but not in complex with factor XI, interacts with the endothelium and can maintain endothelial cell quiescence by preventing endothelial stimulation by thrombin.

The interaction of factor XIa with activated platelets but not endothelial cells promotes the activation of factor IX in the consolidation phase of blood coagulation.

We have previously shown that the zymogen factor XI (FXI) binds to activated platelets but not to human umbilical vein endothelial cells (HUVEC), a conclusion that is in conflict with previous reports stating that FXI binds to 2.7-13 x 10(6) high affinity sites per HUVEC (Berrettini, M., Schleef, R. R., Heeb, M. J., Hopmeier, P., and Griffin, J. H. (1992) J. Biol. Chem. 267, 19833-19839; Shariat-Madar, Z., Mahdi, F., and Schmaier, A. H. (2001) Thromb. Haemostasis 85, 544-551). It has also been reported that activated FXI (FXIa) binds to 1.5 x 10(6) sites per HUVEC and promotes the activation of factor IX by cell bound FXIa (Berrettini, M., Schleef, R. R., Heeb, M. J., Hopmeier, P., and Griffin, J. H. (1992) J. Biol. Chem. 267, 19833-19839). Therefore, the binding of FXIa to activated platelets was compared with FXIa binding to HUVEC and HEK293 cells immobilized on microcarrier beads. Specific and saturable zinc-dependent FXIa binding was demonstrated to 250 +/- 48 sites per activated platelet (K(D) = 1.7 +/- 0.78 nm) and 6.5 +/- 0.4 x 10(4) sites per HUVEC (K(D) = 2.4 +/- 0.5 nm), whereas no binding to HEK293 cells was detected. A titration with high molecular weight kininogen had no effect on FXIa binding to platelets, but revealed a concentration-dependent decrease in the amount of FXIa bound to HUVEC. The rate of factor IXa generation catalyzed by FXIa was unaffected by the presence of surfaces; however only the activated platelet surface protected FXIa from inhibition by protease nexin 2. The results presented here confirm the conclusion that activated platelets are procoagulant while unstimulated endothelial cells are not.

Activated platelets but not endothelial cells participate in the initiation of the consolidation phase of blood coagulation.

To address the question of whether initiation of the consolidation phase of coagulation occurs on platelets or on endothelium, we have examined the interaction of coagulation factor XI with human umbilical vein endothelial cells (HUVEC) and with platelets. In microtiter wells factor XI binds to more sites in the absence of HUVEC (1.8 x 10(10) sites/well, K(D) = 2.6 nm) than in their presence (1.3 x 10(10) sites/well, K(D) = 12 nm) when high molecular weight kininogen (HK) and zinc are present. Binding was volume-dependent and abrogated by HUVEC or Chinese hamster ovary cells and was a function of nonspecific binding of HK to the artificial plastic surface. Factor XI did not bind to HUVEC or to HEK293 cell monolayers anchored to microcarrier beads. Activation of HUVEC resulted in von Willebrand's factor secretion, but factor XI binding was not observed. Only activated platelets supported factor XI binding in the presence of HK and zinc (K(D) = 8 nm, B(max) = 1319 sites/cell). Activation of factor XI was observed in plasma in the presence of platelets activated by the thrombin receptor activation peptide but not with activated HUVEC. These results support the concept that activated platelets, but not endothelial cells, expose a procoagulant surface for binding and activating factor XI, thereby initiating the consolidation phase of coagulation.