SummaryWe determined, in volunteers, the plasma levels of heparin above and below the critical chainlength necessary for thrombin inhibition (ACLM and BCLM), from 1 to 24 h after subcutaneous injection of 5000IU unfractionated heparin (UFH), 40 mg enoxaparin and 1 mg/kg body weight of enoxaparin (LMWH) (n = 12 for each dose). The levels were calculated from the antithrombin- and anti-Xa activities using the specific activities of the materials injected. We also determined the course of thrombin- and of factor Xa generation after triggering the extrinsic system in the same samples. From the thrombin generation curves, we calculated the course of prothrombinase activity.When the ACLM and BCLM plasma-levels are plotted against the inhibition of thrombin- and factor Xa generation, it appears that:a) There is a unique dose response relationship between ACLM level and the inhibition of thrombin generation, independent of whether the ACLM is derived from UFH or LMWH. This relationship is not significantly altered by the BCLM appearing after LMWH injection.b) There is a similar unique relationship between ACLM level and the inhibition of factor Xa generation, again independent of BCLM.c) Inhibition of prothrombin activation hardly contributes to the overall effect on thrombin formation and is again independent of the source of ACLM.d) ACLM levels were significantly higher after injection of LMWH than after UFH injection, even though the amounts of ACLM injected with the highest dose of LMWH were smaller than those administered in the UFH injection.We conclude that the only functional difference between LMWH and UFH is the much higher bioavailability of the former. We surmise that, from the UFH injected, only the lower molecular weight species reach the circulation, i. e. a fraction similar to the ACLM injected with enoxaparin.

von Willebrand factor (vWF) is a multimeric adhesive glycoprotein with one factor VIII binding site/subunit. Prior reports suggest that posttranslational modifications of vWF, including formation of N-terminal intersubunit disulfide bonds and subsequent cleavage of the propeptide, influence availability and/or affinity of factor VIII binding sites. We found that deletion of the vWF propeptide produced a dimeric vWF molecule lacking N-terminal intersubunit disulfide bonds. This molecule bound fluorescein-labeled factor VIII with sixfold lower affinity than multimeric vWF in an equilibrium flow cytometry assay (approximate KDs, 5 nmol/L v 0.9 nmol/L). Coexpression of propeptide-deleted vWF with the vWF propeptide in trans yielded multimeric vWF that displayed increased affinity for factor VIII. Insertion of an alanine residue at the N-terminus of the mature vWF subunit destroyed binding to factor VIII, indicating that the native mature N-terminus is required for factor VIII binding. The requirement for vWF propeptide cleavage was shown by (1) a point mutation of the vWF propeptide cleavage site yielding pro-vWF that was defective in factor VIII binding and (2) correlation between efficiency of intracellular propeptide cleavage and factor VIII binding. Furthermore, in a cell-free system, addition of the propeptide-cleaving enzyme PACE/furin enabled factor VIII binding in parallel with propeptide cleavage. Our results indicate that high-affinity factor VIII binding sites are located on N-terminal disulfide-linked vWF subunits from which the propeptide has been cleaved.

von Willebrand factor (vWf) is a multimeric adhesive glycoprotein that serves as a carrier for factor VIII in plasma. Although each vWf subunit displays a high affinity binding site for factor VIII in vitro, in plasma, only 2% of the vWf sites for factor VIII are occupied. We investigated whether interaction of plasma proteins with vWf or adhesion of vWf to collagen may alter the affinity or availability of factor VIII-binding sites on vWf. When vWf was immobilized on agarose-linked monoclonal antibody, factor VIII bound to vWf with high affinity, and neither the affinity nor binding site availability was influenced by the presence of 50% plasma. Therefore, plasma proteins do not alter the affinity or availability of factor VIII-binding sites. In contrast, when vWf was immobilized on agarose-linked collagen, its affinity for factor VIII was reduced 4-fold, with K D increasing from 0.9 to 3.8 nM. However, one factor VIII-binding site remained available on each vWf subunit. A comparable reduction in affinity for factor VIII was observed when vWf was a constituent of the subendothelial cell matrix and when it was bound to purified type VI collagen. In parallel with the decreased affinity for factor VIII, collagen-bound vWf displayed a 6-fold lower affinity for monoclonal antibody W5-6A, with an epitope composed of residues 78 -96 within the factor VIII-binding motif of vWf. We conclude that collagen induces a conformational change within the factor VIII-binding motif of vWf that lowers the affinity for factor VIII.von Willebrand factor (vWf) 1 is a large multimeric glycoprotein that functions both as an adhesive protein and as a carrier for factor VIII (antihemophilic factor) in plasma. vWf is secreted both from the apical surface of cells into plasma and from the basolateral surface of cells, where it binds to type VI collagen and is incorporated into the vascular matrix (1). As a carrier for factor VIII, plasma vWf functions to prevent factor VIII from binding to phosphatidylserine-containing membranes, where it may be degraded by anticoagulant proteases such as activated protein C (2-4). As an adhesive protein, plasma vWf forms the primary link between exposed extravascular collagen at a site of vascular rupture and platelets, which are sequestered from flowing blood by collagen-bound vWf (5, 6). vWf secreted from the basolateral surface of endothelial cells also functions as an adhesive protein. When the vascular matrix is exposed to flowing blood, the vWf-bound to type VI collagen supports adhesion of platelets and development of a thrombus (7). Factor VIII is released from vWf following proteolytic activation by thrombin (8) and is then free to bind to phosphatidylserine-containing membranes, where it may assemble with the serine protease, factor IXa, to form an efficient procoagulant enzyme complex (9) or be degraded by activated protein C (4). Only suspended, plasma vWf is known to bind factor VIII. Thus, we were motivated to determine the consequences that may occur when circulating vWf with bound factor...

von Willebrand factor (vWF) is a multimeric adhesive glycoprotein with one factor VIII binding site/subunit. Prior reports suggest that posttranslational modifications of vWF, including formation of N-terminal intersubunit disulfide bonds and subsequent cleavage of the propeptide, influence availability and/or affinity of factor VIII binding sites. We found that deletion of the vWF propeptide produced a dimeric vWF molecule lacking N-terminal intersubunit disulfide bonds. This molecule bound fluorescein-labeled factor VIII with sixfold lower affinity than multimeric vWF in an equilibrium flow cytometry assay (approximate KDs, 5 nmol/L v 0.9 nmol/L). Coexpression of propeptide-deleted vWF with the vWF propeptide in trans yielded multimeric vWF that displayed increased affinity for factor VIII. Insertion of an alanine residue at the N-terminus of the mature vWF subunit destroyed binding to factor VIII, indicating that the native mature N-terminus is required for factor VIII binding. The requirement for vWF propeptide cleavage was shown by (1) a point mutation of the vWF propeptide cleavage site yielding pro-vWF that was defective in factor VIII binding and (2) correlation between efficiency of intracellular propeptide cleavage and factor VIII binding. Furthermore, in a cell-free system, addition of the propeptide-cleaving enzyme PACE/furin enabled factor VIII binding in parallel with propeptide cleavage. Our results indicate that high-affinity factor VIII binding sites are located on N-terminal disulfide-linked vWF subunits from which the propeptide has been cleaved.

In determining heparin one has the choice to test a specific activity, such as the decay constant of thrombin or factor Xa on a global test such as the aPTT. The best test would be a global test that directly reflects the only important global effect, to wit antithrombotic efficiency. Such a test does not yet exist. We propose that the thrombin potential, i.e. the time concentration integral of thrombin activity appearing in plasma after triggering is a plausible candidate for such a test.

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