Epitope mapping of the von Willebrand factor subunit distinguishes fragments present in normal and type IIA von Willebrand disease from those generated by plasmin.

Abstract

A small but consistent proportion of the von Willebrand factor (vWF) in normal plasma is composed of 189, 176, and 140 kD fragments cleaved from the 225 kD subunit. A monoclonal antibody map of vWF, based on the reactivity of individual antibodies with cyanogen bromide and tryptic fragments of known carboxy and/or amino termini, showed that in normal and IIA von Willebrand disease (vWD) plasmas the 140 kD fragment was derived from the amino-terminal region, whereas the 176 kD fragment was derived from the carboxy-terminal region of the subunit. In type IIA vWD, however, the fragments comprised a greater proportion of circulating vWF. In contrast, plasmin cleaved a 176 kD fragment from the amino terminus and a 145 kD fragment from the carboxy terminus of the subunit. Species similar to these plasmin-cleaved fragments were demonstrated in plasmas from four patients treated with fibrinolytic agents, but not in IIA vWD.

Plasmin Cleavage of von Willebrand Factor as an Emergency Bypass for ADAMTS13 Deficiency in Thrombotic Microangiopathy

“…The gradual cleavage of globular VWF by plasmin has been reported previously. 7,8 We here propose that the efficiency of VWF cleavage by plasmin is a function of its conformation: Plasmin has limited affinity for the binding to and cleavage of globular VWF, but unfolding of VWF strongly enhances this process. As a result, uPA-triggered plasminogen activation destroyed platelet-VWF complexes on endothelial cells and in suspension in a matter of seconds, which could be recapitulated by streptokinase-activated plasminogen.…”

“…Streptokinase-activated plasminogen gradually degraded purified VWF in solution ( Figure IIIC in the online-only Data Supplement), as reported previously for purified plasmin. 7 Furthermore, streptokinase-activated plasminogen rapidly cleaved platelet-covered VWF strings from endothelial cells under flow ( Figure 3A; Movie VI in the online-only Data Supplement; quantification in Figure 3C). This cleavage was also lysine dependent; εACA protected against streptokinase-mediated degradation of platelet-VWF complexes ( Figure 3B; Movie VII in the online-only Data Supplement).…”

“…Active plasmin is able to cleave VWF and alters its platelet-binding capacity under purified conditions. 7,8 Plasminogen can be activated by tissue-type plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA). Whereas tPA has a high affinity for fibrin, 9 uPA has little affinity for fibrin before activation.…”

Type IIB von Willebrand factor with normal sialic acid content induces platelet aggregation in the absence of ristocetin. Role of platelet activation, fibrinogen, and two distinct membrane receptors.

“…; 5.2% for family P.; and 3.7% for family C. The lower yield of patient vWF is due to the lower efficiency of the cryoprecipitation step, due to the decreased concentration of larger multimers. Analysis of the purified vWF on reduced polyacrylnmide gels (16,17) showed a major band with apparent mobility of 220 kD in both normal and patients and some smaller bands, due to proteolytic degradation, which were increased in the patients (16). All the bands stained by Coomassie Blue were identified as vWF by immunoblotting (17).…”

“…Analysis of the purified vWF on reduced polyacrylnmide gels (16,17) showed a major band with apparent mobility of 220 kD in both normal and patients and some smaller bands, due to proteolytic degradation, which were increased in the patients (16). All the bands stained by Coomassie Blue were identified as vWF by immunoblotting (17). Purified normal and IIB vWF were stored in 0.02 M Tris HCI, 0.15 M NaCl, pH 7.3 (Tris buffer), and kept at -70'C.…”