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Tissue Plasminogen Activator Promotes Platelet Disaggregation in Plasma

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Tissue Plasminogen Activator Promotes Platelet Disaggregation in Plasma Tissue plasminogen activator promotes platelet disaggregation in plasma. J Loscalzo, D E Vaughan J Clin Invest. 1987;79(6):1749-1755. https://doi.org/10.1172/JCI113015. Research Article We studied the disaggregation of human platelets by tissue-type plasminogen activator (t-PA). When added to a suspension of human platelets induced to aggregate in plasma with adenosine 5'-diphosphate, t-PA promoted disaggregation of platelets over several minutes. Addition of fresh plasma or purified human fibrinogen to disaggregated platelets facilitated (reversible) aggregation and subsequent disaggregation. Aspirin treatment of platelets markedly potentiated the ability of t-PA to induce disaggregation. Disaggregation was inhibited by alpha-2-antiplasmin. Comparative analysis of the rate of proteolysis of platelet-bound fibrinogen with that of ambient plasma fibrinogen suggested that fibrinogenolysis of cohesive fibrinogen occurred more rapidly than fibrinogenolysis of ambient fibrinogen. These data demonstrate that t-PA facilitates platelet disaggregation in plasma through kinetically selective proteolysis of cohesive fibrinogen by plasmin, and suggest that thrombolytic mechanisms may serve both to remove platelets from platelet-fibrin thrombi and to disperse circulating platelet aggregates. Find the latest version: https://jci.me/113015/pdf Tissue Plasminogen Activator Promotes Platelet Disaggregation in Plasma Joseph Loscalzo and Douglas E. Vaughan Divisions of Vascular Medicine and Cardiology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115 Abstract local promotion of plasmin production at the platelet surface (7, 10) may also be important in promoting disaggregation of We studied the disaggregation of human platelets by tissue-type platelet aggregates as well as inhibiting aggregate formation. Since plasminogen activator (t-PA). When added to a suspension of platelet aggregates have been implicated in the pathogenesis of human platelets induced to aggregate in plasma with adenosine many clinically important ischemic vascular events (1 1-15), their 5'-diphosphate, t-PA promoted disaggregation of platelets over disaggregation may be a relevant protective mechanism in vivo. several minutes. Addition of fresh plasma or purified human For these reasons, we examined the effect of recombinant t-PA fibrinogen to disaggregated platelets facilitated (reversible) ag- on platelet aggregates produced in vitro in platelet-rich plasma. gregation and subsequent disaggregation. Aspirin treatment of Our results demonstrate that t-PA promotes platelet disaggre- platelets markedly potentiated the ability of t-PA to induce dis- gation in plasma and that its does so through proteolysis of co- aggregation. Disaggregation was inhibited by alpha-2-antiplas- hesive fibrinogen by plasmin. min. Comparative analysis of the rate of proteolysis of platelet- bound fibrinogen with that of ambient plasma fibrinogen sug- gested that fibrinogenolysis of cohesive fibrinogen occurred more Methods rapidly than fibrinogenolysis of ambient fibrinogen. These data demonstrate that t-PA facilitates platelet disaggregation in Materials. Adenosine 5'-diphosphate (ADP), aprotinin, hirudin, and gly- were plasma through kinetically selective proteolysis of cohesive fi- cyl-L-prolyl-L-arginyl-L-proline (GPRP) purchased from Sigma brinogen by plasmin, and suggest that thrombolytic mechanisms Chemical Co., St. Louis, MO. Human fibrinogen, plasmin, plasminogen, streptokinase, and H-D-valyl-L-leucyl-L-lysine-p-nitroanilide may serve both to remove platelets from platelet-fibrin thrombi dihydro- chloride (S-225 1) and di- and to disperse H-D-isoleucyl-L-prolyl-L-arginyl-p-nitroanilide circulating platelet aggregates. hydrochloride (S-2288) were obtained from KABI Diagnostics through Helena Laboratories, Beaumont, TX. Calf skin collagen was purchased Introduction from Cooper Biomedical, Inc., Malvern, PA. Plasminogen-free bovine thrombin was purchased from Miles Pharmaceuticals, Naperville, IL. Tissue-type plasminogen activator (t-PA)' has been intensively Human alpha-2-antiplasmin was obtained from American Diagnostica, studied in recent years as an endogenous biomolecule that is Inc., Greenwich, CT. Recombinant human t-PA was kindly provided important for initiating fibrinolysis (1-5). Plasminogen activation by Genentech, Inc., South San Francisco, CA. Na['251] and Na["31I] were by endogenous t-PA occurs specifically at sites of thrombus for- obtained from New England Nuclear, Boston, MA. Sepharose 2B was mation and fibrin itself enhances this activation (6). Platelets purchased from Pharmacia Fine Chemicals, Inc., Uppsala, Sweden. Iodo- beads were obtained from Pierce Chemical Co., Rockford, IL. have also recently been shown to a provide surface that enhances Collection and preparation of human platelets. Venous blood was t-PA-induced plasminogen activation (7). Once generated, plas- anticoagulated with 13 mM trisodium citrate. Platelet-rich plasma was min not only catalyzes fibrinolysis but also modifies the platelet prepared by centrifugation at 120 g for 10 min at 22°C. The top two- surface, inhibiting platelet aggregation (8, 9). In that thrombi thirds of the platelet-rich plasma was removed for aggregation experi- are composed of fibrin and platelets, and that both the fibrin ments. Platelet counts were determined with a counter equipped with a polymer and platelet surface promote t-PA-mediated plasmin- 50-gm aperture (model ZM; Coulter Electronics Inc., Hialeah, FL). ogen activation, these mechanisms of plasminogen activation Platelets were kept at 22°C for up to 1 h before use. Platelets were treated serve to confine plasmin production and action to the thrombus. with aspirin by incubation for 30 min with 1 mM aspirin at 25°C in platelet-rich plasma. Washed platelets were obtained by While modification of platelets by plasmin inhibits aggre- centrifuging gation in vitro, platelets from platelet-rich plasma at 800 g for 20 min, resuspending the the importance of this mechanism in vivo has platelets in platelet washing buffer (10 mM triosodium citrate, 0.15 M not been established. Furthermore, since fibrinogen, an alter- NaCl, 1 mM ethylenediaminetetraacetate, pH 7.0), centrifuging the native substrate for plasmin, is important in maintaining cohe- platelets at 800 g for 20 min, repeating the suspension in washing buffer sion among platelets induced to aggregate by a variety of agonists, and centrifugation, and finally suspending the platelets at 3.0 X 108/ml in modified Tyrode's solution (0.13 M NaCl, 2.7 mM KCI, 12 mM NaHCO3, 0.42 mM NaH2PO4, pH 7.4, 1.0% bovine serum albumin). Address reprint requests to Dr. Loscalzo, Department of Medicine, Platelet aggregation. Platelets in platelet-rich plasma were typically Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115. suspended at a final concentration of 3.0 X 108/ml and aggregated with Receivedfor publication 31 July 1986 and in revisedform 28 January 1.5 or 11 M ADP, with 0.5 U/ml plasminogen-free bovine thrombin 1987. in the presence of 1 mM GPRP, or with 0.12 mg/ml calfskin collagen. Aggregation (16) was monitored at 370C with stirring at 900 rpm using 1. Abbreviations used in this paper: GPRP, glycyl-L-prolyl-L-arginyl-L- a dual-channel aggregometer (Payton Assoc., Inc., Buffalo, NY). proline; t-PA, tissue-type plasminogen activator. Platelet disaggregation. Disaggregation of platelets in platelet-rich plasma was induced by addition of t-PA after the aggregation response J. Clin. Invest. reached its maximum. Disaggregation was quantitated by measuring ei- © The American Society for Clinical Investigation, Inc. ther (a) the extent of reduction in light transmittance or (b) the maximal 002 1-9738/87/06/1749/07 $ 1.00 rate of decrease in light transmittance after addition of t-PA (in percent Volume 79, June 1987, 1749-1755 transmittance per second). Plasminogen Activator and Platelet Disaggregation 1749 'Platelet radiojodination. Surface proteins of intact platelets were la- aggregation and its extent were dependent on the concentration beled with 1251 using lactoperoxidase (17, 18). The labeled platelets were oft-PA as well as the time of its addition. Higher concentrations suspended in fresh platelet-poor plasma for aggregation and disaggregation oft-PA produced faster rates ofdisaggregation and greater extents experiments. Fresh platelets had a sp act of 1.5 X I07 cpm/109 platelets. of change in light transmittance (Fig. 2). In addition, the earlier Fibrinogen purification. Fibrinogen was purified according to the t-PA was added after ADP, the faster the rate of disaggregation method Bennett and Vilaire (19). Purified fibrinogen was found to be of its extent (Fig. 3). 98% clottable and to contain < 1.5 Ag of fibronectin per 100 ug of protein and the greater and < 1.0 ug ofvon Willebrand factor per'100 fig of protein as determined Reversibility of disaggregation. Once disaggregated, platelets by solid-phase immunoassays using immunoadsorbed polyclonal rabbit appeared normally discoid in shape by phase-contrast micros- anti-human fibronectin and von Willebrand factor antisera, respectively copy. Resuspension of these platelets in fresh platelet-poor (20). The purified fibrinogen was also found to contain < 0.2 zg throm- plasma supported another cycle ofaggregation and disaggregation bospondin per 100 Mg of protein as measured in a radioimmunoas- (Fig. 4). In this experiment, platelets were induced to aggregate say performed by Dr. Henry Slayter, Dana Farber Cancer Institute,
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