ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 15, No. 2 Copyright €> 1985, Institute for Clinical Science, Inc.

Antithrombin III, Plasminogen, , and Alpha-2-Antiplasmin in Donor Blood and Plasma Components

D. K. McROYAN, M.D.,* C. J. McROYAN, MT(ASCP)SH,* K. L. SAUTER, MT(ASCP),* P. I. LIU, M.D., PH.D .,t and S. J. DANIEL, M .D.t

* Hemostasis and Transfusion Divisions, Department of Pathology, University of South Alabama Medical Center tAmerican Red Cross Blood Services, Gulf Coast Region, Mobile, AL 33617

ABSTRACT

The functional levels of antithrombin III, plasminogen, plasmin, and alpha-2 -antiplamsin were evaluated in sequentially derived , cryoprecipitate, and cryo-poor plasma aliquots from 2 0 registered blood donors. Antithrombin III is the major plasma inhibitor of the of the procoagulant system. Plasminogen, the proenzymatic form of plasmin, is the primary endogenous profibrinolytic moiety; alpha- 2-antiplasmin is the principal intermediate acting inhibitor of plasmin. As congenital or acquired deficiencies of antithrombin III and/or plasminogen predispose to , and as these agents may be consumed in acute thrombosis, the goal of this investigation was to discern those plasma components which potentially might maximize both and fibrinolytic activities if used therapeutically. Using enzyme specific synthetic substrate methods, it was determined that no spontaneous plasmin activity was evident through phlebotomy or component processing and storage. Analysis of variance showed antithrom­ bin III to be significantly decreased in cryoprecipitate as compared to the other components (p < 0.0001). Furthermore, the level of antithrombin III or plasminogen in cryo-poor plasma and fresh frozen plasma was not statistically different. Also, the alpha-2-antiplasmin level was not statisti­ cally different among the specimen groups. Since fresh frozen plasma and cryo-poor plasma contain comparable total quantities of antithrombin III and plasminogen and as most of the activity of factors I, V, VIII, and XIII is diverted into cryoprecipitate, it is suggested that cryo-poor plasma may be preferable to fresh frozen plasma for the treatment of thrombosis asso­ ciated with or complicated by antithrombin III and/or plasminogen defi­ ciency.

165 0091-7370/85/0300-0165 $00.90 © Institute for Clinical Science, Inc. 166 McROYAN, McROYAN, SAUTER, LIU, AND DANIEL

Introduction ther, patients with AT-III deficiency unresponsive to oral anticoagulation have A growing number of disorders of been reported . 8,10,16 and have been Plasminogen, the proenzymatic form recognized in recent years which predis­ of plasmin, is the primary mediator of pose to thrombosis. 13,14,19 Among these fibrinolysis. Plasminogen may be con­ thrombotic coagulopathies are deficiency verted by limited to plasmin states involving antithrombin III (AT-III) by a variety of endogenous activators and and plasminogen (PLG). Physiologically, by a number of increasingly important AT-III and PLG are the most important pharmacologic agents . 14 Plasminogen and profibrinolytic moie­ that is affixed to within a ties, respectively. Deficiency states by tissue-type plasminogen activators is involving AT-III or PLG may be congen­ converted to plasmin which acts locally ital or acquired on a chronic or acute and enzymatically releases fibrin degra­ basis. 214,23 Further, superacutely either dation products which are themselves or both may be critically consumed in anticoagulant. Any plasmin which is acute thrombosis . 2 ,3 1 7 Hypothetically, formed free in the circulation is normally thrombosis in the face of either or both rapidly inactivated by alpha-2-antipIas- of these lesions might not respond to min (A2AP), thus delimiting systemic pharmacologic intervention intended for fibrinolysis. 11 Thrombotic coagulopathy anticoagulation or fibrinolysis. ascribable to disorders of the fibrinolytic The cardinal manifestation of a critical system presently appear to be rare. deficiency of AT-III is resistance to hep- However, acute thrombosis may be arinization since is physiologi­ accompanied by a critical deficiency of cally non-functional in the absence of its PLG, hypothetically, leading to a failure cofactor. As the major plasma inhibitor of of endogenous fibrinolysis or thrombo­ the serine proteases of the procoagulant lytic therapy . 3,14 system, AT-III possesses inherent anti­ Blood components or derivatives have activity. This activity, however, rarely been advocated or used as is greatly enhanced and becomes thera­ adjuncts in the treatment of thrombosis peutically meaningful by the binding of or a thrombotic predisposition . 1,2,11,12,13,23 heparin to lysine residues on the AT-III In most cases, AT-III concentrates, molecule. The resultant AT-III:heparin which are investigational and not widely complex both inhibits the formation of available, and fresh frozen plasma have activated serine proteases and rapidly been advocated as sources of AT-III prior inactivates those formed, principally fac­ to heparinization. Mintz et al in 1976 tors Ha, Xa, IXa, and XIa . 20 T he n e t presented evidence to establish both effect of this interaction is that clotting is fresh frozen plasma and cryo-poor plasma inhibited at multiple critical points in the as potential replacement sources of AT- coagulation schema and anticoagulation III . 15 Further, it can be inferred from is effected. It is well known that heparin- other accounts that fresh frozen plasma ization in patients with significant AT-III is a stable source of both AT-III and deficiency is suboptimal; therefore, vita­ PLG . 7,18 However, fresh frozen plasma min K antagonists have been espoused contains significant amounts of factors I, as a means of achieving anticoagulation V, VIII, and XIII, while cryo-poor in these patients. 2,13,23 However, the full plasma contains diminished amounts of antithrombotic effect of oral anticoagu­ these factors which are required for the lants occurs only after a latent period and formation of a stable clot. 24 may not be efficacious acutely. 26,27 F u r­ The effect of thrombosis caused by or ANTITHROMBIN III IN PLASMA COMPONENTS 167 resulting in AT-III and/or PLG defi­ Factor I by activation,* and ciency would be a window of patient vul­ Factors V and VIII utilizing Factors V nerability to rethrombosis and/or poor and VIII deficient plasmat factor assay clot lysis, respectively. The goal of this methods. investigation was to identify those plasma Statistics were analyzed by analysis of components which potentially might variance and paired t-test with signifi­ maximize both antithrombotic and fibri­ cance set at p < 0.05. nolytic activities if therapeutically used as replacement sources of AT-III and/or Results PLG. In table I are shown the results of the Materials and Methods functional assays for AT-III, PLG, and A2AP. As the volume of any component Blood from 20 registered, volunteer may vary considerably from donor to donors was evaluated; written, informed donor, the results and statistical treat­ consent was obtained in each case. ment of these data are expressed on a mil­ Immediate pre- and post-phlebotomy liliter to milliliter basis versus a total vol­ specimens of 4.5 ml each were collected ume basis. 24 The AT-III and A2AP data into buffered sodium citrate in a ratio of are expressed as percent activity per mil­ 9:1, whole blood: citrate. poor liliter of normal human plasma; PLG is plasma was promptly prepared by cen­ expressed as Committee on Thromboly­ trifugation at 1,500 X g for 10 min, ali- tic Activity Units per milliliter of quoted, and stored in polypropylene plasma. 6 tubes at — 70°C. The bank blood was col­ Plasmin activity is not included in the lected in the usual manner into citrate- data as none was found in any specimen phosphate-dextrose-adenine- 1 in a ratio tested. The A2AP level was not statisti­ of 7:1, whole blood: citrate. Fresh frozen cally different among the specimen plasma, cryoprecipitate, and cryo-poor groups. Analysis of variance showed AT- plasma were prepared sequentially from III to be significantly decreased in cryo- each unit collected by conventional precipitate (p < 0 .0 0 0 1 ) as compared to blood bank techniques; aliquots of each the other component. Also, the func­ component were promptly stored in plas­ tional activity of AT-III did not differ sig­ tic tubes at — 70°C.24 nificantly in fresh frozen plasma and The functional AT-III, PLG, plasmin, cryo-poor plasma (p > 0.05). Further, and A2AP activity of each of the five these data suggest that PLG is a partially groups of specimens were assayed using cryoprecipitable plasma and that amidolytic, fluorogen-tagged, enzyme cryoprecipitation results in a modest specific synthetic substrate methods . 6 diversion of PLG into cryoprecipitate. All assays employed Protopath metho­ However, despite this effect, the level of dologies and 5-aminoisophthalic acid PLG is not significantly different in fresh dimethyl ester (AIE) tagged substrates.* frozen plasma and cryo-poor plasma (p > The fluorescent substrates are AT-III, 0.05). D-phe-pro-arg-AIE; PLG and plasmin, Given conventional component pro­ D-val-leu-lys-AIE; and A2AP, D-val-leu- cessing standards, fresh frozen plasma lys-AIE. The aliquots were assayed in must have a minimum volume of 225 mil­ batch and in duplicate within 60 days. In liliters from which a maximum volume of addition, 1 0 aliquots of the sequentially derived fresh frozen plasma and cryo- * American Dade, Miami, FL. poor plasma were assayed in batch for t General Diagnostics; Morris Plains, NY. 168 McROYAN, McROYAN, SAUTER, LIU, AND DANIEL

TABLE I from fresh frozen plasma to cryo-poor Functional Activity of Antithrombin III, plasma (p < 0 .0 0 0 1 ). Plasminogen, and Alpha-2-Antiplasmin in Blood and Components Discussion AT-III PLG A2AP S p e c im e n (%*) (CTA U/ml-f) (%*} The absence of plasmin activity in all of the component groups assayed indi­ P re-ph1ebotomy 100 + 8 3 . 6 ± 0 . 8 116 ± 12 (n = 10) cates that phlebotomy and conventional Post-phlebotomy 98 + 8 3 . 6 ± 0 . 4 116 ± 12 component processing and storage meth­ (n = 10) Fresh frozen plasma 97 + 7 4 . 0 ± 1.0 113 ± 14 ods do not activate the fibrinolytic sys­ (n = 20) tem. This finding suggests that the PLG Cryoprecipitate 75 + 7 6 . 3 ± 1 . 8 121 ± 11 (n = 10) content and, therefore, the potential Cryo-poor plasma 96 + 11 3 .9 ± 1 .2 115 ± 12 (n = 20) fibrinolytic activity of these components is not diminished by present modes of

♦Percent of activity per mL of normal human p l a s m a . preparation. This observation is consis­ fCommittee on Thrombolytic A ctivity Units per ml. . 18 Results expressed as x ± SD. tent with those of Nilsson et al The functional level of AT-III did not vary significantly in fresh frozen plasma 15 milliliters can be diverted into cryo- and cryo-poor plasma. Further, analysis precipitate (precipitable protein plus of variance showed AT-III to be signifi­ supernatant plasma) . 24 The residual vol­ cantly decreased in cryoprecipitate (p < ume expressed after preparation of cryo- 0 .0 0 0 1 ) as compared to fresh frozen or precipitate is cryo-poor plasma and, cryo-poor plasma. This is in contradis­ therefore, represents no more than a tinction to the findings of Mintz et al15; maximum 6.7 percent reduction of the this divergence in findings is undoubtly original fresh frozen plasma volume. On attributable to the greater sensitivity and this basis, the total amount of PLG or specificity of enzyme specific synthetic AT-III per single donor unit of fresh fro­ substrate assays as compared to biologi­ zen plasma or cryo-poor plasma is com­ cal assays. 6 These data suggest that PLG parable. The fresh frozen plasma units is a partially cryoprecipitable plasma pro­ donated to this study had an average vol­ tein which results in a modest diversion ume of 256 ml. Although neither the of PLG into cryoprecipitate and away cryoprecipitate nor cryo-poor plasma vol­ from cryo-poor plasma. The magnitude umes were determined, given the pre- of this effect, however, is not sufficient ceeding standards, an average maximum enough to cause the PLG contents of reduction of 5.9 percent total volume fresh frozen plasma and cryo-poor plasma and, therefore, total activity of PLG and

AT-III would be expected proceeding TABLE I I

from fresh frozen plasma to cryo-poor Function Activity of Factors I, V, and III plasma. in Components In table II are shown the results of the F a c to r I F a c to r V Factor VIII functional assays for factors I, V, and S pecim en (m g/dL) (%*) (%*) VIII. The and factor VIII data are expressed as percent activity per mil­ Fresh frozen plasma 320 ± 67 165 ± 29 151 ± 30 (n = 10) liliter of normal human plasma, and Cryo-poor plasma 193 ± 30 81 ± 16 25 ± 8 is expressed in milligrams per (n = 10) deciliter of plasma. Factors I, V, and VIII ♦Percent of activity per mL of normal human plasma. all showed a highly significant decrease Results expressed as x i SD. ANTITHROMBIN III IN PLASMA COMPONENTS 169

to be significantly different. Therefore, ciated with chronic AT-III deficiency and equal volumes of single donor fresh fro­ thrombosis include hyperestrogenic zen plasma and cryo-poor plasma contain states and the , 9,15 comparable quantities of AT-III or PLG while disseminated intravascular coagu­ (see table I). Also, the total quantity of lopathy and the post-operative state may AT-III or PLG per single donor unit of be associated with an acute defi­ fresh frozen plasma and cryo-poor plasma ciency . 14,17 Further, AT-III and other is comparable (see Results). Further, it coagulation may be consumed is well known that significant amounts of and critically decreased in acute throm­ factors I, V, VIII, and XIII are diverted bosis. 3 from fresh frozen plasma into cryoprecip- Although congenital abnormalities of itate, consequently, rarefying the clotta- PLG are associated with a predisposition bility of cryo-poor plasma; these data are to thrombosis, they presently appear to consistent with a significant diversion of be rare . 14 Acquired hypoplasminogemia Factors I, V, and VIII (see table II). The may be associated with disseminated practical application of these data is that intravascular coagulopathy, postopera­ the potential anticoagulant and fibrino­ tive states, and acute thrombosis. 3,14,22 lytic properties of cryo-poor plasma are Life-threatening thrombosis caused by preserved in processing while its clotta- or resulting in deficiencies of AT-III and/ bility is greatly diminished. This suggests or PLG might not respond to therapeutic that cryo-poor plasma may be a valuable intervention intended for anticoagulation adjunct in the treatment of thrombosis or fibrinolysis. As AT-III concentrates associated with AT-III and/or PLG defi­ are investigational and not widely avail­ ciency. able, fresh frozen plasma has been These data suggest that cryoprecipi- espoused as a source of AT-III. 13,22,23 This tate is an enriched source of PLG. Cryo- approach to replacement therapy might precipitate as a source of PLG cannot be be more effective if cryo-poor plasma recommended because the total units of were used. These data indicate that cryo- PLG activity per single donor unit are poor plasma contains quantities of AT-III low. Achieving a meaningful therapeutic and PLG comparable to fresh frozen dose of PLG would probably necessitate plasma. Further, as a result of processing the use of multiple units which might the clottability of cryo-poor plasma is rar­ expose the recipient to an inordinately efied by the diversion of factors I, V, high hepatitis risk . 24 Also, the fibrinolytic VIII, and XIII into cryoprecipitate, potential of cryoprecipitate may be whereas fresh frozen plasma is not. A negated by its thrombogenic potential. model is currently being developed to Congenital and acquired deficiencies test the efficacy of cryo-poor plasma in of AT-III are associated with a predis­ thrombosis. position to thrombosis. It is suggested that AT-III levels between 50 to 75 per­ cent of normal confer a moderate risk for thrombosis, and levels less than 50 per­ References cent indicate a significant risk . 2,6,23 Con­ 1. Bernhardt, W. and N o v a k o v a , A.: Antithrombin genital AT-III deficiency is the most III concentrates in intensive care. La Ricerca common known heritable state associ­ Clin Lab i3:61-66, 1983. ated with thrombosis and may be as 2. B i c k , R. L.: Clinical relevance of antithrombin III. Sem. Thromb. Haemost. 8:276-287, 1982. prevalent as one per 2000 to 5000.13 3. B i c k , R. L., and M c C l a i n , B . J.: Deep venous Acquired conditions which may be asso­ thrombosis: A laboratory evaluation of 118 con­ 170 McROYAN, McROYAN, SAUTER, LIU, AND DANIEL

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