Characterization of the von Willebrand factor present in circulating PMN

MAUGERI NORMA; KEMPFER ANA C; SAVINO JESSICA; POZNER, ROBERTO; NEGROTO, SOLEDAD; AMARAL MARÍA M; SCHATTNER, MIRTA; LAZZARI MARÍA A

Birmingham

Congreso; XIX CONGRESS. THE INTERNATIONAL SOCIETY ON THROMBOSIS AND HAEMOSTASIS; 2003

THE INTERNATIONAL SOCIETY ON THROMBOSIS AND HAEMOSTASIS

We have previously reported the presence of von Willebrand factor (VWF) on PMN surface, which acts as a bridge between platelets and PMN. It has been recently shown that Mac-1 is the receptor to the immobilized form of VWF, such as the present in vascular subendothelium. The purpose of the present work was to characterize VWF present on PMN. Study of multimeric pattern of VWF, showed that VWF of leukocytes only have the small multimers (n = 8). Lysates of PMN or platelets were analyzed by reducing and nonreducing SDS-9% PAGE, blotted and probed with an antibody against VWF, followed by immunoenzymatic stain and evaluated by densitometric tracing (n = 6). Contrarily, to the nonreduction pattern of solubilized platelets, VWF bound to PMN showed only two bands of 80 and c. 30 kDa while the pattern of VWF on PMN samples in reducing conditions yielded a single band of c. 30 kDa. A similar proteolytic pattern was found after electrophoresis under nonreducing conditions of a recombinant VWF that only contains A1-A2-A3 domains. The possibility that structural characteristics of leukocyte VWF would be due to the proteases derived from PMN was also considered and analyzed. Purified VWF was coincubated with PMN stimulated with 1 muM of fMLP in the presence of cytochalasin B (2.5 mug mL-1 ) at 37 °C for 10 min and Western blot was performed as described above. Results showed that the exposure of VWF to proteases derived from PMN did not generate the VWF pattern of 80 and 30 kDa observed in leukocyte VWF. In order to elucidate when VWF is incorporated to PMN surface, we analyzed its presence in PMN precursors (isolated from human bone marrow). Flow cytometry studies showed that VWF was completely absent in PMN precursors (22 ± 12 mean ± SEM of anti-VWF fluorescence, n = 4) as compared with circulating (mature) PMN (221 ± 28, mean ± SEM of anti-VWF fluorescence, n = 8, P < 0.01). Simultaneously determination of Mac-1 demonstrated that receptor of VWF on PMN is expressed in precursors (285 ± 32 mean ± SEM of anti-CD11b fluorescence, n = 4) and circulating (567 ± 26 mean ± SEM of anti-CD11b fluorescence, n = 8) PMN. In conclusion, our observations indicate that the VWF present on PMN (1) have very low molecular weight (2) probably have a dimeric structure (3) this structure is not due to the effect of protease derived from PMN and (4) is incorporate to PMN after maturation, probably during the trans-endothelial migration from bone marrow to the circulation.