Thrombin-stimulated human platelets express molecular forms of alpha-granule factor V (FV), which differ from FVa.
Binding of 125I-Fab fragments of chain-specific antibodies indicate that both heavy and light chains of alpha-granule factor Va (FVa) were externalized on the platelet membrane after stimulation with thrombin. Using a Mab against the activation peptide of factor V (FV), the epitope appears on the stimulated platelet surface. Half as much light chain and heavy chain (FVa) was expressed compared to the activation peptide, suggesting that expression of alpha-granule FV occurs after thrombin stimulation. Using an ELISA, we find that 32% of alpha-granule FV was released and 68% is retained in the platelet pellet. Immunoblots of platelets indicate that FV exists in 200 kDa und 150 kDa forms, representing incomplete cleavage, while the releasate demonstrates a more complete cleavage by proteases. We conclude that expression of alpha-granule FV is quantitatively greater than that released and exists in molecular forms which cannot be completely explained by the binding of FVa.
Wyshock
E G
EG
Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140.
Stewart
G J
GJ
Colman
R W
RW
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D004797:Enzyme-Linked Immunosorbent Assay; D005165:Factor V / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D015943:Factor Va / Q000032:analysis* / Q000737:chemistry; D006801:Humans; D008081:Liposomes; D008565:Membrane Proteins / Q000097:blood* / Q000737:chemistry; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D013268:Stimulation, Chemical; D013917:Thrombin / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D008081:Liposomes; D008565:Membrane Proteins; D015943:Factor Va; D005165:Factor V; D013917:Thrombin