Megakaryocyte proplatelet-like process formation in vitro is inhibited by serum prothrombin, a process which is blocked by matrix-bound glycosaminoglycans.
The process of platelet shedding from megakaryocytes is incompletely understood, due in part to the impossibility of studying this dynamic process in vivo. Megakaryocytes in situ and in in vitro cultures display extended cytoplasmic processes constricted at platelet-sized intervals which presumably are the structural intermediates between megakaryocytes and platelets. This study describes the establishment of a serum-free culture system of purified guinea pig megakaryocytes in which extensive cytoplasmic process formation can be observed on 21 to 29% of the cells. The addition of as little as 0.05% pooled human serum to the cultures will completely but reversibly block process development. The serum inhibitor was identified as residual prothrombin, which upon contact with megakaryocytes is converted to the serine esterase thrombin. Thrombin directly prevents the formation of new processes and also induces retraction of existing processes. When megakaryocytes are cultured on Matrigel, process formation occurs even in an excess of thrombin. This potentiation of process development in the presence of inhibitory factors is mediated by the glycosaminoglycan content of Matrigel. The physiological implications of these observations are discussed.
Hunt
P
P
Laboratory of Stem Cell Biology, Amgen Center, Thousand Oaks, CA 91320.
Hokom
M M
MM
Wiemann
B
B
Leven
R M
RM
Arakawa
T
T
D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D001798:Blood Proteins / Q000494:pharmacology; D002478:Cells, Cultured; D002850:Chromatography, Gel; D002918:Chymotrypsin / Q000494:pharmacology; D003094:Collagen; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004338:Drug Combinations; D004347:Drug Interactions; D004950:Esterases / Q000494:pharmacology; D006025:Glycosaminoglycans / Q000494:pharmacology*; D006168:Guinea Pigs; D006497:Heparan Sulfate / Q000494:pharmacology; D007797:Laminin; D008533:Megakaryocytes / Q000166:cytology* / Q000187:drug effects; D011509:Proteoglycans; D011516:Prothrombin / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D013917:Thrombin / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D001798:Blood Proteins; D002918:Chymotrypsin; D003094:Collagen; D016895:Culture Media, Serum-Free; D004338:Drug Combinations; D004950:Esterases; D006025:Glycosaminoglycans; D006497:Heparan Sulfate; D007797:Laminin; D011509:Proteoglycans; D011516:Prothrombin; D013917:Thrombin; D014357:Trypsin; C059485:matrigel; C022635:serine esterase