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Blood clotting in minimally altered whole blood
MD Rand, JB Lock, C van't Veer, DP Gaffney and KG Mann
Department of Biochemistry, University of Vermont College of Medicine,
Burlington 05405-0068, USA.
The sequences of events regulating thrombin generation during tissue
factor-initiated clotting in whole blood at 37 degrees C in which the
contact pathway was suppressed with corn trypsin inhibitor are studied
using quantitative Western blotting of factor V, prothrombin, platelet
factor 4, antithrombin III, and fibrinogen. In addition, fibrinopeptide A
(FPA), thrombin-antithrombin III (TAT) complex formation, and prothrombin
fragment 1.2 (F1.2) were measured via commercially available enzyme-linked
immunosorbent assays (ELISAs). In a typical experiment initiated with 40
pmol/L recombinant tissue factor, visual clot time (4.5 minutes), was
preceded by significant cleavage of factor V resulting in 65% factor Va
heavy-chain generation but only 10% light- chain formation. At this point,
50% of the platelet factor 4 is released, suggesting that half
(approximately 700 pmol/L) of the platelet prothrombinase sites available
have been generated. At clot time, approximately 15 nmol/L thrombin B-chain
is present; however, analyses of FPA release demonstrate that only 15% of
the thrombin is acting on fibrinogen. This thrombin is produced by the
action of 7 pmol/L prothrombinase. The maximum rate of thrombin production
is reached well after clot time and is consistent with the presence of
approximately 150 pmol/L prothrombinase (at about 7 minutes). These results
suggest that factor Xa is the limiting factor for thrombin generation.
After 60 minutes, 75% of the initial prothrombin (1.24 mumol/L) is consumed
yielding 400 nmol/ L prethrombin 2 and 360 nmol/l thrombin (B-chain)
products. The sum of these values (800 nmol/L) is similar to the
(corrected) F1.2 concentration determined by ELISA. The incomplete cleavage
of prothrombin indicates both the prothrombinase complex and the formation
of prothrombinase are inhibited in the reaction. TAT complex measured by
ELISA is almost equivalent to B-chain concentration, but sodium dodecyl
sulfate stable thrombin-antithrombin III complexes are not observed until
well after clot formation and are never equivalent to ELISA-TAT values. At
the point of clot formation, 80% of the fibrinogen is depleted from the
fluid phase, whereas only 35% to 45% of the FPA is released, suggesting a
significant incorporation of uncleaved fibrinogen into the initial clot
formed.
Volume 88,
Issue 9,
pp. 3432-3445,
11/01/1996
Copyright © 1996 by The American Society of Hematology
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