Blood coagulation kinetics: high throughput method for real-time reaction monitoring

Ken Lo; Scott L. Diamond

doi:10.1160/TH04-03-0154

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2004; 92(04): 874-882
DOI: 10.1160/TH04-03-0154

New Technologies and Diagnostic Tools

Schattauer GmbH

Blood coagulation kinetics: high throughput method for real-time reaction monitoring

Ken Lo

¹Department of Chemical and Biomolecular Engineering, Institute of Medicine and Engineering, University of Pennsylvania, Vagelos Research Laboratories, Philadelphia, USA

,

Scott L. Diamond

¹Department of Chemical and Biomolecular Engineering, Institute of Medicine and Engineering, University of Pennsylvania, Vagelos Research Laboratories, Philadelphia, USA

› Author Affiliations

Abstract

Summary

A high throughput 384-well plate assay of blood function in 60 μl reactions with the fluorogenic thrombin substrate, boc-VPR-MCA, allowed for real-time monitoring of coagulation under a diverse set of reaction conditions. Using recalcified, citrated whole blood diluted 3-fold with corn trypsin inhibitor (to block Factor XIIa), addition of 0 to 13.8 pM of tissue factor (TF) reduced the time of maximal rate of thrombin production (T_max) from 45 min to 11 min. Over this range of TF,T_max was reduced from 35 min to 6 min by co-addition of 10 nM convulxin to activate platelets via GPVI. The maximal rate of thrombin production at T_max was not a function of exogenously-added TF, Va, or reVIIa, but increased 30% with added convulxin. Addition of 0.07 to 0.7 pM TF along with convulxin produced small, but detectable reductions in T_max. Addition of up to 0.67 nM reVIIa reduced T_max by up to 53% in the range of 0.7 to 7 pM TF. Interestingly, platelet factor 4 (2.7 μM) caused a prolongation of T_max from 45 min to 78 min at 0 TF, while protamine (1.8 μM) reduced T_max to 30 min at 0 TF. Finally, combinatorial reaction studies with exogenously-added ADP, histamine, fMLP, indomethacin, anti-CD18, and fibrinogen revealed no unusual synergies amongst the agents, but demonstrated a striking procoagulant activity of added fibrinogen, due to protease contaminants in the “purified” fibrinogen. This high throughput approach allowed automated profiling of blood (50 reactions/ml of blood) to generate large data sets for testing cellular-proteomic kinetic models, screening drug interactions, and potentially monitoring subtle changes in the functional phenotype of a patient blood sample.

Keywords

Fluorescence - thrombin - kinetics - platelet factor 4

Full Text

References

References
1 Eerkes A, Shou WZ, Naidong W. Liquid/liquid extraction using 96-well plate format in conjunction with hydrophilic interaction liquid chromatography - tandem mass spectrometry method for the analysis of fluconazole in human plasma. J Pharm Biomed Anal 2003; 31: 917-28.
2 Xie F, Bruntlett CS, Zhu Y. et al. Good preclinical bioanalytical chemistry requires proper sampling from laboratory animals: automation of blood and microdialysis sampling improves the productivity of LC/MSMS. Anal Sci 2003; 19: 479-85.
3 Sugimoto M, Matsui H, Mizuno T. et al. Mural thrombus generation in type 2A and 2B von Willebrand disease under flow conditions. Blood 2003; 101: 915-20.
4 Klein B, Faridi A, von Tempelhoff GF. et al. A whole blood flow cytometric determination of platelet activation by unfractionated and low molecular weight heparin in vitro. Thromb Res 2003; 108: 291-6.
5 Gosalia DN, Diamond SL. Printing chemical libraries on microarrays for fluid phase nanoliter reactions. Proc Natl Acad Sci U S A 2003; 100: 8721-6.
6 Hockin MF, Jones KC, Everse SJ. et al. A model for the stoichiometric regulation of blood coagulation. JBC 2002; 277: 18322-33.
7 Kuharsky AL, Fogelson AL. Surface-mediated control of blood coagulation: the role of binding site densities and platelet deposition. Biophys J 2001; 80: 1050-74.
8 Bungay SD, Gentry PA, Gentry RD. A mathematical model of lipid-mediated thrombin generation. Math Med Biol 2003; 20: 105-29.
9 Doggett TA, Girdhar G, Lawshe A. Alterations in the intrinsic properties of the GPIbalphaVWF tether bond define the kinetics of the platelet-type von Willebrand disease mutation, Gly233Val. Blood 2003; 102: 152-60.
10 Laurenzi IJ, Diamond SL. Monte Carlo simulation of the heterotypic aggregation kinetics of platelets and neutrophils. Biophys J 1999; 77: 1733-46.
11 Butenas S, van’t CVeer, Mann KG. Evaluation of the initiation phase of blood coagulation using ultrasensitive assays for serine proteases. JBC 1997; 272: 21527-33.
12 Van’t CVeer, Mann KG. Regulation of tissue factor initiated thrombin generation by the stoichiometric inhibitors tissue factor pathway inhibitor, antithrombin-III, and heparin cofactor-II. JBC 1997; 272: 4367-77.
13 Gailani D, Broze GJ. Factor XI activation in a revised model of blood coagulation. Science 1991; 253: 909-12.
14 Brummel-Ziedins KE, Pouliot RL, Mann KG. Thrombin generation: phenotypic quantitation. J Thromb Haemost 2004; 02: 281-8.
15 Butenas S, Brummel KE, Branda RF. et al. Mechanism of factor VIIa-dependent coagulation in hemophilia blood. Blood 2002; 99: 923-30.
16 Hemker HC, Giesen P, Al Dieri R. et al. Calibrated automated thrombin generation measurement in clotting plasma. Pathophysiol Haemost Thromb 2003; 33: 4-15.
17 Hemker HC, Giesen PL, Ramjee M. et al. The thrombogram: monitoring thrombin generation in platelet-rich plasma. Thromb Haemost 2000; 83: 589-91.
18 Hemker HC, Giesen P, Al Dieri R. et al. The calibrated automated thrombogram (CAT): a universal routine test for hyper- and hypocoagulability. Pathophysiol Haemost Thromb 2002; 32: 249-53.
19 Holmes MB, Schneider DJ, Hayes MG. et al. Novel, bedside, tissue factor-dependent clotting assay permits improved assessment of combination antithrombotic and antiplatelet therapy. Circulation 2000; 102: 2051-7.
20 Polgár J, Clemetson JM, Kehrel BE. et al. Platelet activation and signal transduction by convulxin, a C-type lectin from Crotalus durissus terrificus (tropical rattlesnake) venom via the p62/GPVI collagen receptor. JBC 1997; 272: 13576-83.
21 Camire RM, Kalafatis M, Simioni P. et al. Platelet-derived factor Va/Va Leiden cofactor activities are sustained on the surface of activated platelets despite the presence of activated protein C. Blood 1998; 91: 2818-29.
22 Tracy PB, Eide LL, Bowie EJ. et al. Radioimmunoassay of factor V in human plasma and platelets. Blood 1982; 60: 59-63.
23 Lloyd JV, Joist JH. Recombinant factor VIIa: a universal hemostatic agent?. Curr Hematol Rep 2002; 01: 19-26.
24 Kaplan KL, Owen J. Plasma levels of betathromboglobulin and platelet factor 4 as indices of platelet activation in vivo. Blood 1981; 57: 199-202.
25 Bruins P, te HVelthuis, Eerenberg-Belmer AJ. et al. Heparin-protamine complexes and Creactive protein induce activation of the classical complement pathway: studies in patients undergoing cardiac surgery and in vitro. Thromb Haemost 2000; 84: 237-43.
26 Rauch U, Nemerson Y. Circulating tissue factor and thrombosis. Curr Opin Hematol 2000; 07: 273-7.
27 Goel MS, Diamond SL. Factor VIIa-mediated tenase function on activated platelets under flow. J Thromb Haemost 2004; 08: 1402-10.
28 Brummel KE, Paradis SG, Butenas S. et al. Thrombin functions during tissue factor-induced blood coagulation. Blood 2002; 100: 148-52.
29 Lowe GD. The relationship between infection, inflammation, and cardiovascular disease: an overview. Ann Periodontol 2001; 06: 1-8.
30 Weitz JI. A novel approach to thrombin inhibition. Thromb Res 2003; 109: S17-22.