326 A factor from endothelium facilitates activation of plasminogen by tissue plasminogen activator

326 A factor from endothelium facilitates activation of plasminogen by tissue plasminogen activator

141 326A FACTOR FROM ENDOTHELIUM FACILITATES ACTIVATION OF PLASMINOGEN BY TISSUE RASMINOGEN ACTIVATOR R. Machovich and W.G. Owen Section of Hematol...

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141

326A FACTOR FROM ENDOTHELIUM FACILITATES

ACTIVATION

OF PLASMINOGEN BY TISSUE RASMINOGEN ACTIVATOR R. Machovich and W.G. Owen Section of Hematology Research, Mayo Clinic/ Foundation, Rochester, Minnesota 55905, U.S.A. The soecificities of the various cofactors for facilitating plasminogen activation suggest roles in catabolism of fibrin and denatured extracellular protein and in the maintenance of a vascular surface free of deposits of insoluble protein. The latter function would predict a capacity of the endothelial cell membrane to regulate plasminogen activation. We extracted and partially purified a component with tPA cofactor activity from porcine coronary endothelium by perfusing Langendorff swine heart

Cofactor preparations. extracted from cultured endothelial

in extracts

cells. This of platelets,

activity porcine activity

The activity

was bound to DE-trisacryl, from which it by increased ionic strength. Like

leukocytes. quantitatively was eluted fibrin(ogen)

fragments, the activity of this The inhibited by 6-aminohexanoate. mechanism of action of endothelial cell cofactor differs from that of others. because its activity can be accounted fir as increasing Vmax without significant effect on Km. It is concluded that endothelium contains a biochemically unique cofactor for tPA.

cofactor

is

was also aortic valve was not

erythrocytes,

found

or

327INFLUENCE OF RETRACTIONON THE LYSABILITY OF LABORATORY CLOTS M. SaboviE, D. Keber Diversity Institute of Gerontology Internal Clinic Tmovo, Ljubljana, Yugoslavia The influence of retraction and preincubation in plasma on the lysability of clots with different agents (streptokinase, urokinase, tissue plasminogen activator:mt-PA) was studied in an "in vitrotl system. Each experiment was performed on whole blood clots from 10 healthy donors. After pretreatment (different degrees of retraction, preincubation in plasma) clots of approximately 300 mg were hanged in 2 ml of plasma or buffered saline with addition of lytic agent (SK: 500 IU/ml, UK 500 IU/ml, t-PA 250 IU/ml). Degree of lysis after 24 hours was expressed as percent decrease of original weight. Nearly all nonretracted clots lysed completely both in saline and titrated plasma, but they did not lyse after homogenization in tissue mixer. Retracted clots

PLASMZNOGEN ACTIVATION BY FIBRIN-BOUND 328 tPA IN A PLASMA ENVIRONMENT : ROLE OF ALPHA-2-ANTZPLASMZN. D. Rouy and E. Angles-Cano ZNSERM U. 143, Hdpital Bice^tre, Parts, Fmnce.

In the preser&$tudy the kinetics of the activation of native and Z-glu-plasminogen (glu-Pg) by fibrinbound tPA were analysed in the absence and presence of ourified aloha-2antiolasmin ( ~r2-AP. a kind aift of Dr. ‘H.R. L’ijnen) and ‘in plasma using a solid-ph‘h$e fibrin support. tPA (concentration varying from 5 to 20 Iv/ml) was allowed to bind to the solid-phase fibrin plate. Then 100 ul per well of a satumting amount of $&Pg (400 nM) containing a trace amount (1 nMZ of Z-alu-Pa and suoolemented or not with 200 nM ‘(Y2-Ap was” added. ‘Zn pamllel experiments ~~t~~~~~~~M~~~~~e~2~-~~u~~~o gg; . the activation of plasminogen, aliquots of supernatant as well as of the products present on or eluted from the fibrin surface were analysed at regular

lysed neglibly in saline (x,(s)): UK=3,9% (8,5), t-PA=7,1% (10,3); in plasma they SK=7178 (8,8), lysed better only with t-PA: 53,1% (25,3), but not so with UK: 15% (12,O) or SK: 20,6% (13,4). After 24 hours of incubation in plasma, clots lysed excellently both in plasma: uK=90,4% (17,3), X=94,4% (13,1), t-PA=98,6% (4,3) and in saline: UK=90,9% (16,2), SK=99,3% (2,2), t-PA= Lysability of incubated clots was 99,4x (1,2). good even after homogenization. Homogenation of unretracted clots Conclusions: abolished lysis of remaining fibrin in saline, demonstrating that the amount of plasminogen bound to fibrin during coagulation was not sufRetraction had the same efficient for lysis. fect. Better lysis of well retracted clots in plasma with t-PA in contrast to UK and SK can be explained by binding of plasminogen to clots in presence of t-PA. Good lysis of retracted clots after preincubation in plasma, even when homogenized, suggested additional plasminogen binding to fibrin after retraction.

intervals by SDS-PAGE and autoradiogmphy, and by spectrophotometry using a plasmin-selective chromogenic substmte. The amount of fibrin-bound plasmin(ogen) was estimated by counting the mdioactivity of the wells in a gamma counter. Without a2-AP the amount of plasmin genemted increased with time in both the solid-phase and the liquidphase. In the presence of a2-AP, the amount of fibrin-bound plasmin increased with time but to a lesser extent, while no plasmin activity was detected in the liquid-phase. Similar results were obtained using human plasma. In the absence of CY2-AP, bound plasmin was identified to be exclusively of the glutype while in the supernatant both glu- and lys-type were present. In the presence of (Y 2-AP and in plasma, only glu-plasmin was identified. These results suggest that 1) glu-plasmin is the main fibrinolytic enzyme and that lys-plasminogen is not an intermediary product of the activation in plasma, 2) (r2-AP does not inhibit the genemtion of plasmin by fibrinbound tPA.