Thromsbospondin-1 binds to the heavy chain of elastase activated coagulation factor V (FVaHNE) and enhances thrombin generation on the surface of a promyelocytic cell line.
BACKGROUND
Thrombospondin 1 (TSP1) has the ability to bind to HL-60 cells and to reversibly inhibit human neutrophil elastase (HNE). Human factor V (FV) can be cleaved by HNE thereby providing FV with cofactor activity (FVa(HNE)). Experiments were performed to evaluate the ability of HNE expressed on the surface of HL-60 cells to generate FVa(HNE) to support thrombin generation, and to determine the effect of TSP1 on this reaction.


RESULTS
Western blot analysis showed TSP1 forming a complex with FVa(HNE) within a region corresponding to the heavy chain of FV. Enzymatic reactions were performed to determine the role of TSP1-HNE-FVa(HNE) on the surface of HL-60 cells, namely the assembly of the prothrombinase complex. Thrombin generation was measured by the chromogenic substrate S2238. Exposure of factor V to HL-60 cells prior to the addition of prothrombin and activated factor X provided FV with cofactor activity. HL-60 cells were found capable of synthesizing factor V with cofactor activity, but HL-60 cells failed to synthesize and/or to provide factor X with enzymatic activity. The ability of HL-60 cells to synthesize FV and TSP1 was demonstrated. The addition of exogenous TSP1 enhanced both the rate and amount of thrombin generated on the HL-60 cell surface.


CONCLUSIONS
Despite the ability of TSP1 to reversibly inhibit HNE in a purified system, TSP1 expression favors the reactions leading to thrombin generation on the HL-60 cell surface. These observations are relevant to clinical conditions where there is a prothrombotic state such as malignant tumors.
Isordia-Salas
Irma
I
Temple University School of Medicine, Department of Physiology, The Sol Sherry Thrombosis Research Center, 3400 North Broad Street Philadelphia, PA 19140, USA.
Manns
Joanne M
JM
Sainz
Irma
I
Parekh
Hemant
H
DeLa Cadena
Raul A
RA
D015153:Blotting, Western; D004789:Enzyme Activation; D005165:Factor V / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D019272:Leukocyte Elastase / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D013917:Thrombin / Q000096:biosynthesis*; D019700:Thrombospondin 1 / Q000378:metabolism*
D012333:RNA, Messenger; D019700:Thrombospondin 1; D005165:Factor V; D005170:Factor X; D019272:Leukocyte Elastase; D013917:Thrombin