Further studies on acid-labile [32P]phosphate bound to high-molecular weight material from rat-liver cell sap after incubation with [32P]adenosine triphosphate

Gel filtration of human plasma cryoprecipitate on Sepharose 2B indicated the molecular weight of factor VIII coagulant activity (VIIIc) to be significantly greater than that found in antihaemophilic concentrate. Polyethylene glycol at 3% concentration precipitated approximately half of the VIIIc from cryoprecipitate. This activity eluted as high molecular weight material on gel filtration. The addition of more polyethylene glycol to a concentration of 8% precipitated most of the remaining VIIIc from cryoprecipitate. This activity appeared to be of significantly lower molecular weight, approximately corresponding in elution volume to that observed for antihaemophilic concentrate. The possibility that an antibody to VIIIc generated in a patient treated with cryoprecipitate might be directed against the higher molecular weight form of factor VIII was investigated. However, no significant differences between the higher and lower molecular weight forms of factor VIII either in stability or in reactivity with human antibody to factor VIII were found.

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Platelet protein organization: analysis by treatment with membrane- permeable cross-linking reagents

Blood ◽

10.1182/blood.v59.3.502.502 ◽

1982 ◽

Vol 59 (3) ◽

pp. 502-513 ◽

Cited By ~ 36

Author(s):

GE Davies ◽

J Palek

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Polyacrylamide Gel Electrophoresis ◽

Cytoskeletal Proteins ◽

Cross Linking ◽

Membrane Glycoproteins ◽

Platelet Protein ◽

High Molecular Weight Material ◽

Organization Analysis ◽

Thrombin Activation

Abstract We have examined platelet protein organization by treatment of intact resting or thrombin-activated platelets with two cross-linking reagents, diamide or dithiobis(succinimidyl propionate) (DTSP). Cross- linked complexes were separated by polyacrylamide gel electrophoresis in the absence of reducing agent and their composition determined after reductive cleavage and analysis in a second-dimensional gel. The most prominent cross-linked species produced by diamide treatment of of resting platelets are (A) cytoskeletal protein homopolymers, such as myosin heavy chain dimer and actin oligomers, and (B) high molecular weight material consisting of homo- or heteropolymers of cytoskeletal proteins and 230,000, 170,000, 100,000, 55,000, and 52,000 dalton proteins. DTSP treatment forms similar complexes and also cross-links membrane glycoproteins IIb and III into high molecular weight material. Thrombin activation of platelets before treatment with diamide or DTSP results in increased cross-linking of myosin and increased incorporation of several proteins, particularly myosin and glycoproteins IIb and III, into high molecular weight material. The results provide evidence for reorganization of cytoskeletal and membrane proteins during platelet function.

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