Prostaglandin D₂(PGD₂) synthesis in activated mast cells occurs in two phases, an early phase that is dependent on prostaglandin synthase 1 and a delayed phase that is dependent on activation-induced prostaglandin synthase 2 gene expression. Early phase PGD₂synthesis in activated mast cells also requires the activity of a secretory phospholipase A₂ (PLA₂). It has been thought that the secretory PLA₂ expressed in mast cells is group IIa PLA₂, encoded by the Pla2 g2a gene. However, activated bone marrow-derived mast cells prepared fromPla2 g2a ^+/+ mice and mast cells prepared from mice with a mutation in the Pla2 g2a gene both demonstrate early phase PGD₂ synthesis. Moreover, mast cells from both murine strains secrete PLA₂ activity following activation. Northern and reverse transcriptase/polymerase chain reaction analyses demonstrate that mast cells from Pla2 g2a ^+/+and Pla2 g2a ^−/− mice do notexpress group IIa PLA₂ message. Instead, Northern and reverse transcriptase/polymerase chain reaction analyses demonstrate that both Pla2 g2a ^+/+ and Pla2 g2a ^−/− mast cells express mRNA for group V PLA₂, encoded by the Pla2gV gene. An antisense oligonucleotide directed against group V PLA₂ is also able to inhibit both the early phase of PGD₂ production and the secretion of PLA₂ activity by activated mast cells. Our data suggest that (i) group IIa PLA₂ does not play a significant role in murine mast cell prostaglandin synthesis, (ii) group V PLA₂ mediates early mast cell PGD₂production and transcellular PGE₂ production in murine mast cells, and (iii) much of the data, based on studies with chemical inhibitors and antibodies, suggesting that group IIa PLA₂is responsible for arachidonic acid mobilization needs to be reevaluated.

We describe the use of phage libraries to derive new antibodies against p21Ras to be used for intracellular expression in mammalian cells. A panel of single‐chain antibody fragments, binding to Ras, were analyzed and characterized for their capacity to interfere in vitro with (a) the intrinsic GTPase activity of Ras and (b) the binding of Ras to its effector Raf, and were found not to neutralize its function, according to these biochemical criteria. When expressed intracellularly in mouse 3T3 K‐Ras transformed cells all the anti‐Ras single‐chain variable fragments (scFv) tested inhibited cell proliferation, as assessed by bromodeoxyuridine incorporation. Double immunofluorescence analysis of transfected cells using confocal microscopy confirmed that anti‐Ras antibody fragments colocalize with endogenous Ras, at subcellular locations where the protein Ras is not normally found. These data suggest that the ability of phage‐derived anti‐Ras scFv fragments to inhibit the function of Ras in vivo is a rather general and frequent property and that the range of antibodies that can be successfully used for intracellular inhibition studies is much greater than anticipated, exploiting the mode of action of diverting protein traffic.

The low density lipoprotein receptor-related protein (LRP), a multifunctional endocytic receptor, mediates the cellular internalization of tissue-type (t-PA) and urokinase-type (u-PA) plasminogen activator and their complexes with plasminogen activator inhibitor type 1 (PAI-1). LRP preferentially binds the complexed forms, exemplified by equilibrium dissociation constants (K_D) that are at least an order of magnitude lower than those of the free components. To understand the molecular interactions, underlying the preference of the receptor for complexes rather than for the free components, we have performed a detailed analysis of the affinity and kinetics of the binding of PAI-1 and t-PA:PAI-1 complexes to the receptor, using surface plasmon resonance. To assess the involvement of the heparin-binding domain of PAI-1 for the interaction with LRP, we determined the equilibrium dissociation constants for the binding to LRP of a panel of PAI-1 mutants with single- and multiple amino-acid substitutions of the basic residues that constitute the heparin binding site of PAI-1 (K65, K69, R76, K80 and K88). The binding of these PAI-1 mutants was partially reduced with a 2 to 4 fold increase in K_D values for single (K80, K88) and combined (K80, 88) substitution mutant proteins respectively. LRP binding of complexes, composed of t-PA with either wild type PAI-1 or any one of the single PAI-1 mutants indicated a major role of lysine 69 (K69) for the binding of t-PA:PAI-1 complexes to LRP (K_D values of 6.1, 3.7, 75.4, 5.4, 12.5 and 8.1 nM for wild type, K65A, K69A, R76A, K80A and K88A complexes, respectively). Since the K_D for the binding of free t-PA to LRP is 158 nM, we conclude that the PAI-1 moiety harbors the major determinant for t-PA:PAI-1 complex binding to LRP. The in vitro binding studies were extended by binding and clearance studies with COS-1 cells. Degradation of both ¹²⁵I-t-PA:PAI-1 K69A and ¹²⁵I-t-PA:PAI-1 K69A K80A K88A complexes after 2 h of incubation was reduced compared to the degradation of ¹²⁵I-t-PA:PAI-1 complexes. We conclude that PAI-1 contains a cryptic binding site (lysine 69) for LRP, that is specifically expressed upon t-PA:PAI-1 complex formation. ^* This work was supported by grant no. 902-26-128 from the Netherlands Organization for Scientific Research (NWO). A.-J.v.Z. is a recipient of a fellowship from the Royal Netherlands Academy of Arts and Sciences (KNAW). ^** Essential parts of this study have been reported at the XIIIth International Congress on Fibrinolysis and Thrombolysis (Barcelona, Spain June 24–28, 1996) and published in Fibrinolysis 10 (suppl. 3) (1996) abstr. 62.