- Email: [email protected]
91. Factor Va plays a major role in determining the fibrinolytic potential of a clot
28
Fibrinolysis
and 0.46 for the deletion allele, and were in Hardy Weinberg equilibrium. Homozygosity for the insertion (n=138) was associated with an increased risk of myocardial infarction compared to homozygosity (n=75) for the deletion: relative risk 2.0 (95% CI 1.1, 3.6). Conclusion: These findings suggest an association of the insertion allele of the t-PA gene with the occurrence of myocardial infarction. The I/D polymorphism seems to be a linkage marker for an unknown mutation at, or near, the tPA gene. The insertion allele may reflect an impaired capacity of the fibrinolytic system to respond adequately to acute coronary thrombosis.
90. A p r o t i n i n a n d its effects o n c h r o m o g e n i c substrate assays for some components of the plasma defence systems H. P. Wendel, W. Heller, H.-M. Hoffmeister
University of Tuebingen, Depts. of Cardiovascular Surgery and Internal Medicine III*, 72076 Tuebingen, Germany Aprotinin, a proteinase inhibitor from bovine lungs is increasingly being used at high doses in cardiac surgery to
reduce blood loss. Whilst studying samples from patients undergoing cardiopulmonary bypass (CPB), we observed that whenever aprotinin was infused, marked changes in levels of some of the components we were measuring occurred. We suspected that aprotinin was affecting the chromogenic substrate (CS) assays we were using. We have therefore studied the effects of aprotinin on CS assays for plasminogen (PEG), alpha2-antiplasmin (alpha2-AP), tissue plasminogen activator (tPA), plasminogen activator inhibitor (PAI), prekallikrein (PKK) and kallikrein inhibition (KKI). Aprotinin was added to citrated human plasma to give final concentrations of from 0-400 KIU/ml and the plasma samples assayed for PLG, alpha2-AP, PAI, PKK and KKI. For the tPA assay, aprotinin was added to blood from an exercised donor before acidification and assay. We observed significant apparent falls in levels of PLG, tPA and PKK and significant apparent increases in alpha2-AP, PAI and KKI. These changes were dependent on aprotinin concentration. Similar effects were seen in samples from CPB patients undergoing high dose aprotinin therapy. Our results show that interpretation of results from chromogenic substrate assays on samples containing proteinase inhibitors must take into consideration the effects of the inhibitors on these assays.
OTHER HAEMOSTATIC RISK MARKERS 91. F a c t o r Va p l a y s a major role in d e t e r m i n i n g the fibrinolytic p o t e n t i a l o f a clot L. Bajzar 1, M. Kalafatis 1, P. Simioni 2, M. E. N e s h e i m 3, P. B. T r a c y 1
1University of Vermont, USA, 2University of Padua, Italy 3Queens University, Canada The importance of a thrombin activatable fibrinolysis inhibitor (TAFI) on tPA-induced fibrinolysis of clots formed from purified components has been previously described. Upon activation, TAFI exhibits carboxypeptidase B-like exopeptidase specificity. Since, C-terminal lysines of partially degraded fibrin are considered to be the most potent cofactor for tPA-induced activation of plasminogen, it is hypothesized that their removal by activated TAFI inhibits fibrinolysis. Since, TAFI is activated by thrombin we propose that regulation of thrombin formation will regulate fibrinolysis in a TAFI-dependent manner. A key step might include regulation of the concentration of factor Va. Physiologically this would include down regulation by proteolytic inactivation by activated protein C (APC) or up regulation by secretion of factor Va from platelets. Using a quantitative turbidometric lysis assay we showed that APC (ECso=5 nM) shortens lysis time by 3-fold only in clots formed under conditions where prothrombinase-dependent formation of thrombin can occur. However, its EC5o is increased to 50 nM when its effect on clot lysis is determined using plasma from patients who are homozygous for the factor VL e i d e n mutation. This mutation renders the factor Va less susceptible to inactivation by APC. Under conditions where prothrombin was activated, in a non-prothrombinase dependent manner, APC was unable to shorten lysis time. In addition, APC did not Fibrinolysis (1996) 10, Suppl. 1, 1-58
shorten lysis time of clots formed from plasma immunodepleted of TAFI, however, the effect could be reconstituted with purified TAFI. These data indicate that APC appears profibrinolytic, in a TAFI-dependent manner, by regulating factor Va-dependent activation of prothrombin, exclusively. The inclusion of platelets in plasma prolonged lysis time in a manner that was attentuated by both APC and active site blocked factor Xa. However, platelets reduced the potency of APC (EC5o=20 nM). APC was also able to attenuate plateletdependent prolongation of lysis time of clots formed from factor V-deficient plasma, though no APC effect was observed in the plasma in the absence of platelets. Addition of platelets to TAFI-deficient plasma did not prolong lysis time. These data indicate that platelets appear antifibrinolytic by secretion of platelet factor Va, thereby, potentiating the TAFI-dependent inhibition of fibrinolysis. Thus, factor Va provided by either plasma or platelet stores can be considered an important regulator of fibrinolysis.
92. Lp(a) l i p o p r o t e i n a n d r i s k o f atherosderotic/thrombotic disease K. Berg
Insitute of Medical Genetics, University of Oslo, P.O.B. 1036 Blindern, N-0135 Oslo, Norway The genetically determined Lp(a) lipoprotein whose specific apoprotein evolved from plasminogen exhibits association with atherosclerotic/thrombotic diseases including coronary heart disease (CHD). People with an Lp(a) lipoprotein concentration corresponding to the top quartile in the healthy population have a significantly increased CHD risk compared with © Pearson Professional Ltd 1996
Fibrinolysis
and 0.46 for the deletion allele, and were in Hardy Weinberg equilibrium. Homozygosity for the insertion (n=138) was associated with an increased risk of myocardial infarction compared to homozygosity (n=75) for the deletion: relative risk 2.0 (95% CI 1.1, 3.6). Conclusion: These findings suggest an association of the insertion allele of the t-PA gene with the occurrence of myocardial infarction. The I/D polymorphism seems to be a linkage marker for an unknown mutation at, or near, the tPA gene. The insertion allele may reflect an impaired capacity of the fibrinolytic system to respond adequately to acute coronary thrombosis.
90. A p r o t i n i n a n d its effects o n c h r o m o g e n i c substrate assays for some components of the plasma defence systems H. P. Wendel, W. Heller, H.-M. Hoffmeister
University of Tuebingen, Depts. of Cardiovascular Surgery and Internal Medicine III*, 72076 Tuebingen, Germany Aprotinin, a proteinase inhibitor from bovine lungs is increasingly being used at high doses in cardiac surgery to
reduce blood loss. Whilst studying samples from patients undergoing cardiopulmonary bypass (CPB), we observed that whenever aprotinin was infused, marked changes in levels of some of the components we were measuring occurred. We suspected that aprotinin was affecting the chromogenic substrate (CS) assays we were using. We have therefore studied the effects of aprotinin on CS assays for plasminogen (PEG), alpha2-antiplasmin (alpha2-AP), tissue plasminogen activator (tPA), plasminogen activator inhibitor (PAI), prekallikrein (PKK) and kallikrein inhibition (KKI). Aprotinin was added to citrated human plasma to give final concentrations of from 0-400 KIU/ml and the plasma samples assayed for PLG, alpha2-AP, PAI, PKK and KKI. For the tPA assay, aprotinin was added to blood from an exercised donor before acidification and assay. We observed significant apparent falls in levels of PLG, tPA and PKK and significant apparent increases in alpha2-AP, PAI and KKI. These changes were dependent on aprotinin concentration. Similar effects were seen in samples from CPB patients undergoing high dose aprotinin therapy. Our results show that interpretation of results from chromogenic substrate assays on samples containing proteinase inhibitors must take into consideration the effects of the inhibitors on these assays.
OTHER HAEMOSTATIC RISK MARKERS 91. F a c t o r Va p l a y s a major role in d e t e r m i n i n g the fibrinolytic p o t e n t i a l o f a clot L. Bajzar 1, M. Kalafatis 1, P. Simioni 2, M. E. N e s h e i m 3, P. B. T r a c y 1
1University of Vermont, USA, 2University of Padua, Italy 3Queens University, Canada The importance of a thrombin activatable fibrinolysis inhibitor (TAFI) on tPA-induced fibrinolysis of clots formed from purified components has been previously described. Upon activation, TAFI exhibits carboxypeptidase B-like exopeptidase specificity. Since, C-terminal lysines of partially degraded fibrin are considered to be the most potent cofactor for tPA-induced activation of plasminogen, it is hypothesized that their removal by activated TAFI inhibits fibrinolysis. Since, TAFI is activated by thrombin we propose that regulation of thrombin formation will regulate fibrinolysis in a TAFI-dependent manner. A key step might include regulation of the concentration of factor Va. Physiologically this would include down regulation by proteolytic inactivation by activated protein C (APC) or up regulation by secretion of factor Va from platelets. Using a quantitative turbidometric lysis assay we showed that APC (ECso=5 nM) shortens lysis time by 3-fold only in clots formed under conditions where prothrombinase-dependent formation of thrombin can occur. However, its EC5o is increased to 50 nM when its effect on clot lysis is determined using plasma from patients who are homozygous for the factor VL e i d e n mutation. This mutation renders the factor Va less susceptible to inactivation by APC. Under conditions where prothrombin was activated, in a non-prothrombinase dependent manner, APC was unable to shorten lysis time. In addition, APC did not Fibrinolysis (1996) 10, Suppl. 1, 1-58
shorten lysis time of clots formed from plasma immunodepleted of TAFI, however, the effect could be reconstituted with purified TAFI. These data indicate that APC appears profibrinolytic, in a TAFI-dependent manner, by regulating factor Va-dependent activation of prothrombin, exclusively. The inclusion of platelets in plasma prolonged lysis time in a manner that was attentuated by both APC and active site blocked factor Xa. However, platelets reduced the potency of APC (EC5o=20 nM). APC was also able to attenuate plateletdependent prolongation of lysis time of clots formed from factor V-deficient plasma, though no APC effect was observed in the plasma in the absence of platelets. Addition of platelets to TAFI-deficient plasma did not prolong lysis time. These data indicate that platelets appear antifibrinolytic by secretion of platelet factor Va, thereby, potentiating the TAFI-dependent inhibition of fibrinolysis. Thus, factor Va provided by either plasma or platelet stores can be considered an important regulator of fibrinolysis.
92. Lp(a) l i p o p r o t e i n a n d r i s k o f atherosderotic/thrombotic disease K. Berg
Insitute of Medical Genetics, University of Oslo, P.O.B. 1036 Blindern, N-0135 Oslo, Norway The genetically determined Lp(a) lipoprotein whose specific apoprotein evolved from plasminogen exhibits association with atherosclerotic/thrombotic diseases including coronary heart disease (CHD). People with an Lp(a) lipoprotein concentration corresponding to the top quartile in the healthy population have a significantly increased CHD risk compared with © Pearson Professional Ltd 1996