We report three members of a family who had reduced levels of plasma von Willebrand factor (vWF) and increased ristocetin-induced platelet aggregation (RIPA) (aggregation of platelet-rich plasma with ristocetin at a concentration of 0.45 mg/mL), as previously reported in type IIB and pseudo-von Willebrand's disease (vWD). However, in contrast to the latter two disorders in which the larger vWF multimers are absent in plasma, the entire range of vWF multimers was observed in the patients' plasma after sodium dodecyl sulfate-agarose gel electrophoresis, and all vWF multimers (including the largest) were present in the same proportion as in normal plasma and type I vWD. Thus, despite increased RIPA, the levels and multimeric pattern of vWF in this family's plasma were indistinguishable from those in type I vWD in which RIPA is usually decreased. Addition of ristocetin to the patients' platelet- rich plasma resulted in the removal of vWF (and, more selectively, of the large multimers) at lower concentrations of ristocetin than normal, as in type IIB and pseudo-vWD. The defect in the patients was localized to their vWF, which had an enhanced capacity for aggregating washed normal platelets in the presence of low concentrations of ristocetin and for aggregating pseudo-vWD platelets (in the absence of ristocetin). Both glycoproteins (GP) Ib and IIb-IIIa were involved in the enhanced aggregation response. RIPA (at low ristocetin concentrations) in the patients' platelet-rich plasma was abolished by a monoclonal antibody (AP1) to GPIb and was markedly reduced by monoclonal antibodies (10E5 and LJP9) that block adenosine diphosphate and thrombin-induced binding of vWF and fibrinogen to GPIIb-IIIa but was unaffected by an antibody (LJP5) that only blocks vWF binding. Partial inhibition of the initial aggregation slope (and complete inhibition of second phase aggregation) was achieved with creatine phosphate/creatine phosphokinase. EDTA blocked second-phase aggregation but was without effect on the initial slope. The findings in this family combine some features of both type I vWD (normal pattern of vWF multimers in plasma) and type IIB vWD (increased RIPA) and further demonstrate the increasing complexity of the structure-function relationships in vWD.
Factor-VIII-related antigen has previously been shown to be synthesized by vascular endothelial cells. Using both an immunofluorescent staining technique and electron microscopy, we have demonstrated the presence of factor-VIII-related antigen in human vascular subendothelium. This finding may have implications in the mechanism of platelet adhesion to deendothelialized blood vessel surfaces.
The multimeric composition of human endothelial cell (EC)-derived von Willebrand factor (vWF) was studied using SDS-agarose gel electrophoresis and autoradiography. Two multimers were found in lysates prepared from confluent cultures of human umbilical vein endothelial cells. The smaller multimer had a molecular weight (mol wt) of approximately 950 Kd, while the second was larger than those seen in plasma. When electrophoresis was performed using the discontinuous buffer system of Ruggeri and Zimmerman, the small multimer consisted of a single band migrating with the slowest-moving component of the corresponding plasma triplet. The large EC-vWF multimer was detected in culture media conditioned with EC monolayers for ten minutes. It remained the only multimer in media conditioned for up to three days. Calcium ionophore A23187 increased the amount of the large vWF multimer released into the culture media, but did not change its multimeric composition. The small multimer was never detected in the EC- conditioned media. These findings suggest that (1) a large, fully polymerized multimer of vWF is released from the ECs, while the small multimer probably represents a major intermediate component in the process of multimerization, and (2) plasma vWF multimers are probably generated from the large endothelial vWF after it is released into the circulation.
The interaction of red blood cells (RBCs) with vascular endothelium under flow conditions was investigated using the perfused rat mesocecum. Under videomicroscopy, normal human erythrocytes were found to adhere to the venular endothelium of desmopressin-treated microvasculature. Transmission electron microscopy showed that the erythrocytes were attached to the endothelial cells at discrete electron-dense sites. Compared with control preparations in which the microvasculature was perfused with Ringer's-albumin solution alone, more than a 10-fold increase in radioactivity was retained in the desmopressin-treated microvasculature when technetium (99mTc)-labeled erythrocytes were infused into the vasculature. This erythrocyte adherence was accompanied by a higher increment in vascular resistance during the passage of RBCs through the microcirculation, and by a delay in the recovery toward baseline. The erythrocyte adherence in desmopressin-treated microvasculature was completely abolished with antibodies to von Willebrand factor (vWF). Desmopressin infusion in rats resulted in elevated vWF antigen levels and the appearance of extra-large molecular weight forms of vWF in plasma. These findings suggest that normal erythrocytes adhere to desmopressin-conditioned microvascular endothelium and that endothelial cell-derived vWF is involved in the erythrocyte-endothelium interaction.
We have localized factor-VIII-related antigen, using immunofluorescence and electron microscopy, in adult human blood vessels. In addition to its presence in endothelial cells, the antigen was localized within subendothelium and the layers of elastic lamina closest to the lumen. Also, we provide the first morphological evidence that factor-VIII- related antigen is associated with collagen fibrils within the vessel wall. These studies suggest that this subendothelial factor-VIII- related antigen may play a role in the adhesion of platelets to subendothelial components following endothelial injury.
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