BACKGROUND: Thrombi are known to induce activation of the coagulation system, which may be a mechanism for progression of thrombosis and its recurrence after thrombolysis. This study was designed to characterize the relative role of thrombin and activated factor X (factor Xa) as mediators of procoagulant activity of whole blood clots in blood and plasma. METHODS AND RESULTS: Clots formed from human blood were incubated in recalcified (25 mmol/L CaCl2) citrated plasma or nonanticoagulated blood with increasing concentrations of recombinant desulfatohirudin (hirudin) to inhibit thrombin activity, recombinant tick anticoagulant peptide (TAP) or recombinant tissue factor pathway inhibitor (TFPI) to inhibit factor Xa, or heparin. Fibrinopeptide A (FPA) was assayed serially as an index of procoagulant (thrombin) activity. FPA generation was greatly accelerated by addition of clots to recalcified plasma (from 1251 +/- 211 ng/mL after 15 minutes without clot to 5916 +/- 1412 ng/mL with clot, n = 7, P < .01) or whole blood (4803 +/- 761 ng/mL with clot compared with 546 +/- 233 without clot, n = 5, P < .05) and was attenuated by inhibitors of thrombin (> 90% inhibition of FPA with 0.05 mumol/L hirudin and 1.0 U/mL heparin) and factor Xa (> 90% inhibition of FPA with 1.0 mumol/L TAP and 0.15 mumol/L TFPI) in a concentration-dependent manner. Preincubation of clots with tissue-type plasminogen activator sufficient to induce partial clot lysis increased the rate of thrombin-induced FPA generation by increasing the surface area of clot exposed to plasma. However, procoagulant activity induced by partially lysed clots was attenuated by lower concentrations of both thrombin and Xa inhibitors, presumably because access of the inhibitors to bound procoagulant molecules was facilitated. Comparable results were obtained with incubations in nonanticoagulated blood. CONCLUSIONS: These results indicate that factor Xa is primarily responsible for the procoagulant activity of clots in vitro and suggest a potential molecular mechanism for the observed efficacy of inhibitors of factor Xa in preventing recurrent thrombosis after coronary thrombolysis.
BACKGROUND: Thrombi are known to induce activation of the coagulation system, which may be a mechanism for progression of thrombosis and its recurrence after thrombolysis. This study was designed to characterize the relative role of thrombin and activated factor X (factor Xa) as mediators of procoagulant activity of whole blood clots in blood and plasma. METHODS AND RESULTS: Clots formed from human blood were incubated in recalcified (25 mmol/L CaCl2) citrated plasma or nonanticoagulated blood with increasing concentrations of recombinant desulfatohirudin (hirudin) to inhibit thrombin activity, recombinant tick anticoagulant peptide (TAP) or recombinant tissue factor pathway inhibitor (TFPI) to inhibit factor Xa, or heparin. Fibrinopeptide A (FPA) was assayed serially as an index of procoagulant (thrombin) activity. FPA generation was greatly accelerated by addition of clots to recalcified plasma (from 1251 +/- 211 ng/mL after 15 minutes without clot to 5916 +/- 1412 ng/mL with clot, n = 7, P < .01) or whole blood (4803 +/- 761 ng/mL with clot compared with 546 +/- 233 without clot, n = 5, P < .05) and was attenuated by inhibitors of thrombin (> 90% inhibition of FPA with 0.05 mumol/L hirudin and 1.0 U/mL heparin) and factor Xa (> 90% inhibition of FPA with 1.0 mumol/L TAP and 0.15 mumol/L TFPI) in a concentration-dependent manner. Preincubation of clots with tissue-type plasminogen activator sufficient to induce partial clot lysis increased the rate of thrombin-induced FPA generation by increasing the surface area of clot exposed to plasma. However, procoagulant activity induced by partially lysed clots was attenuated by lower concentrations of both thrombin and Xa inhibitors, presumably because access of the inhibitors to bound procoagulant molecules was facilitated. Comparable results were obtained with incubations in nonanticoagulated blood. CONCLUSIONS: These results indicate that factor Xa is primarily responsible for the procoagulant activity of clots in vitro and suggest a potential molecular mechanism for the observed efficacy of inhibitors of factor Xa in preventing recurrent thrombosis after coronary thrombolysis.
Authors: Keren Borensztajn; Jurriën Stiekema; Sebastiaan Nijmeijer; Pieter H Reitsma; Maikel P Peppelenbosch; C Arnold Spek Journal: Am J Pathol Date: 2008-01-17 Impact factor: 4.307
Authors: Hao Fan; John J Irwin; Benjamin M Webb; Gerhard Klebe; Brian K Shoichet; Andrej Sali Journal: J Chem Inf Model Date: 2009-11 Impact factor: 4.956