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H Pannekoek

Publications and source records attributed to H Pannekoek.

At least 91 records · Page 5Linked to original sources
PubMed1989

Interaction between plasminogen activator inhibitor type 1 (PAI-1) bound to fibrin and either tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA). Binding of t-PA/PAI-1 complexes to fibrin mediated by both the finger and the kringle-2 domain of t-PA.

Plasminogen activation is catalyzed both by tissue-type-(t-PA) and by urokinase-type plasminogen activator (u-PA). This reaction is controlled by plasminogen activator inhibitor type 1 (PAI-1) that is either present in plasma or bound to fibrin, present in a thrombus. We studied the mechanism of in vitro inhibition of both t-PA and u-PA activity by PAI-1 bound to fibrin. It is shown that activation of latent PAI-1 unmasks a specific fibrin-binding site that is distinct from its reactive site. This reactive site of activated PAI-1 bound to fibrin is fully exposed to form complexes with t-PA and u-PA, that are unable to activate plasminogen. Upon complex formation with either one of the plasminogen activators, PAI-1 apparently undergoes a conformational change and loses its affinity for fibrin. Consequently, complexes of u-PA and PAI-1 dissociate from the fibrin matrix and are encountered in the fluid phase. In contrast, t-PA/PAI-1 complexes remain bound to fibrin. By employing recombinant t-PA deletion-mutant proteins, that precisely lack domains involved in fibrin binding, we demonstrate that binding of t-PA/PAI-1 complexes is mediated by both the "finger" (F) and the "kringle-2" (K2) domain of t-PA. A model is proposed that explains inhibition of the fibrinolytic process, at the level of plasminogen activation by t-PA, directed by PAI-1 bound to fibrin. An implication of the proposed model is that t-PA/PAI-1 complexes and free t-PA compete for the same binding sites on fibrin.

Animals↗
PubMed1988

Carrier detection in severe (type III) von Willebrand disease using two intragenic restriction fragment length polymorphisms.

DNA from a family with a female member affected with severe (type III) vWD was analysed using three restriction enzymes and a partial vWF cDNA probe. Two restriction fragment length polymorphisms (RFLPs) detected with the enzymes Bgl II and Xba I proved to be informative in this family. A 36.0 Kb allele demonstrated with the enzyme Xba I was rare in the general population but very important in this family for segregation analysis of the alleles and their association with the putative defective chromosome. The propositus was homozygous for the 36.0 Kb Xba I polymorphic band and heterozygous for the Bgl II polymorphism. She was the only member of the family showing this allelic pattern. The linkage of the alleles could be determined because her mother was homozygous for the 9.0 Kb Bgl II polymorphism but heterozygous for the Xba I polymorphism. The segregation of the alleles could be traced to the proband's son and a niece. The genotypic analysis revealed that her niece could be considered as carrying a defective gene for severe vWD.

Alleles↗
PubMed1988

Proteolytic cleavage of the precursor of von Willebrand factor is not essential for multimer formation.

Monkey kidney cells (COS-1), transfected with full-length human von Willebrand factor (vWF) cDNA encoding the precursor of vWF (pro-vWF), mimic the characteristics of the biosynthesis and of the constitutive secretory pathway, displayed by cultured vascular endothelial cells. Such heterologous transfected cells are able to cleave pro-vWF, generating the propolypeptide and mature vWF, and to assemble pro-vWF dimers into a series of multimers, similarly to endothelial cells. Evidence is presented showing that proteolytic processing of pro-vWF by COS-1 cells occurs at the peptide bond between arginine and serine in the sequence Lys762-Arg763-Ser764, identical to endothelial cell-associated proteolysis. This conclusion stems from the observation that substitution of Arg763 by a glycine residue completely abolishes proteolytic processing. As a result, transfection of COS-1 with the mutant vWF-Gly763 cDNA does not significantly affect the multimeric organization of secreted vWF molecules. Consequently, we conclude that proteolytic processing of pro-vWF is not required for multimer formation. Pulse-chase labeling of COS-1 cells transfected with full-length vWF cDNA reveals pro-vWF exclusively in cell lysates, whereas both pro-vWF and mature vWF are encountered in the conditioned medium. These observations indicate that proteolytic processing of pro-vWF is a "late" event during intracellular routing of these molecules or may occur extracellularly.

Animals↗
PubMed1988

Artificial exon shuffling between tissue-type plasminogen activator (t-PA) and urokinase (u-PA): a comparative study on the fibrinolytic properties of t-PA/u-PA hybrid proteins.

We constructed two human tissue-type plasminogen activator/urokinase (t-PA/u-PA) hybrid cDNAs which were expressed by transfection of mouse Ltk- cells. The properties of the secreted proteins were compared with those of recombinant t-PA (rt-PA) and high molecular weight (HMW) u-PA. The hybrid proteins each contain the amino-terminal fibrin-binding chain of t-PA fused to the carboxy-terminal serine protease moiety of u-PA but differ by a stretch of 13 amino acid residues between kringle 2 of t-PA and the plasmin cleavage site of u-PA. Hybrid protein rt-PA/u-PA I contains amino acids 1-262 of t-PA connected with amino acids 147-411 of u-PA, whereas hybrid protein rt-PA/u-PA II consists of the same t-PA segment and residues 134-411 of u-PA. We demonstrated fibrin binding for rt-PA, whereas the hybrid proteins bind to a lesser extent and HMW u-PA has no affinity for fibrin. Plasminogen activation by either one of the hybrid proteins in the absence of a fibrin substitute was similar to that by HMW u-PA, while rt-PA was much less active. The catalytic efficiency, in the presence of a fibrin substitute, increases more than 2000-fold for rt-PA, about 250-fold for hybrid proteins I and II, and 12-fold for HMW u-PA, respectively. Under these conditions the hybrid proteins are more efficient plasminogen activators than the parental ones. The hybrid molecules form a 1:1 molar complex with the human endothelial plasminogen activator inhibitor (PAI-1), analogous to that formed by rt-PA and HMW u-PA. The relative affinity of rt-PA for PAI-1 is 4.6-fold higher than that of HMW u-PA.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

Genetic linkage of two intragenic restriction fragment length polymorphisms with von Willebrand's disease type IIA. Evidence for a defect in the von Willebrand factor gene.

Restriction fragment length polymorphisms (RFLPs), using the enzymes Bgl II and Xba I in conjunction with human von Willebrand factor (vWF) cDNA probes, have been described previously. In the present study we demonstrate the localization of both genetic markers within the vWF gene. The RFLPs were used to study the segregation of alleles associated with von Willebrand's disease (vWD) type IIA in a comprehensive, affected family. Individuals of this family were tested for their bleeding time and their plasma was analyzed for vWF antigen concentration and vWF ristocetin-cofactor activity. Based on these data, the affected members were diagnosed as vWD type-IIA patients; this conclusion was confirmed by the analysis of the multimeric vWF pattern of some of the patients. It was demonstrated that both RFLPs are completely linked with the vWD type-IIA trait. From this finding, we conclude that the defect that causes the vWD type IIA is most likely due to a mutation in the vWF gene and not to a mutation in a gene involved in posttranslational processing of the vWF protein.

Chromosome Mapping↗
PubMed1987

Activation of human endothelial cell-type plasminogen activator inhibitor (PAI-1) by negatively charged phospholipids.

The endothelial cell-type plasminogen activator inhibitor (PAI-1) may exist in an inactive, latent form that can be converted into an active form upon treatment of the protein with denaturants, such as sodium dodecyl sulfate, guanidine HCl, or urea. The present paper demonstrates that latent PAI-1 can be activated by lipid vesicles containing the negatively charged phospholipids phosphatidylserine (PS) or phosphatidylinositol. The presence of a net negative charge on the phospholipid headgroup is essential for activation, since lipid vesicles consisting exclusively of zwitterionic phospholipids, such as phosphatidylcholine and phosphatidylethanolamine, do not activate PAI-1. In the presence of PS vesicles, PAI-1 inhibited tissue-type plasminogen activator 50-fold more effectively than in the absence of phospholipids, whereas sodium dodecyl sulfate enhanced PAI-1 activity by 25-fold. In mixed phospholipid vesicles containing PS and phosphatidylcholine in various molar ratios, the extent of PAI-1 activation was directly related to the PS content of the phospholipid membrane. Ca2+ ions interfered with the inhibitory activity of PS-activated PAI-1, suggesting that Ca2+ ions may regulate PAI-1 activity in the presence of negatively charged phospholipids. An important consequence of these findings is that, as in blood coagulation, negatively charged phospholipids may play an important regulatory role in controlling the fibrinolytic system by activating an inhibitor of tissue-type plasminogen activator.

Calcium Chloride↗
PubMed1987

Molecular cloning and expression of hybridoma growth factor in Escherichia coli.

Human monocytes produce a factor that supports the growth of B lymphocyte hybridoma cells, termed hybridoma growth factor (HGF). By using expression cloning in Escherichia coli of complementary DNA derived from human monocyte-poly(A+) RNA, we selected seven clones producing HGF activity as measured in a bioassay, based on the induction of proliferation of the HGF-dependent B cell hybridoma B9. Sequence analysis of the cDNA revealed that HGF is identical with interferon-beta 2, 26,000 protein, and B cell stimulatory factor-2. One of the active clones contained a cDNA that encoded a recombinant product lacking the 28-amino acid long signal peptide and the first 15 amino acids of the mature protein. Antibodies against the recombinant HGF inhibited the biologic activity of recombinant HGF as well as of monocyte-derived HGF.

Amino Acid Sequence↗
PubMed1987

Structure of the human plasminogen activator inhibitor 1 gene: nonrandom distribution of introns.

Plasminogen activator inhibitor 1 (PAI-1) is the primary inhibitor of tissue-type plasminogen activator and thus performs an essential role in the regulation of the fibrinolytic process. It is a member of a large family of serine protease inhibitors (serpins). We determined the structure of the PAI-1 gene in order to more completely investigate the relationship of PAI-1 to other serpins and, at the same time, to begin to delineate structure-function relations in PAI-1 itself. A human genomic cosmid DNA library was screened and found to contain two independent clones, each harboring the entire PAI-1 gene. Restriction site mapping, electron microscopic inspection of heteroduplexes, and nucleotide sequence analysis all demonstrate that the PAI-1 gene is approximately 12.2 kilobase pairs in length and consists of nine exons and eight introns. All intron-exon boundaries are in accord with the "GT-AG" rule, including a cryptic acceptor splice site found in intron 7. The intron-exon pattern of the PAI-1 gene is distinct from that of most other serpins except that intron 3 of PAI-1 occupies an identical position as intron E of ovalbumin. Comparison of our data with the proposed subdomain structure of serpins suggests that seven of the eight introns may occupy a nonrandom position in the gene. These introns either delineate boundaries of individual structural subdomains or are located in random coil regions of the protein. Transcription of the PAI-1 gene in cultured vascular endothelial cells results in two distinct mRNA species. Our data suggest that these two transcripts arise by alternative polyadenylation.

Base Sequence↗
PubMed1987

Mapping of epitopes on human tissue-type plasminogen activator with recombinant deletion mutant proteins.

An antigen assay based on a monoclonal antibody directed against the light chain of tissue-type plasminogen activator (t-PA) was developed to quantify seven recombinant (r) t-PA deletion mutant proteins. These recombinant proteins were then employed to map different epitopes on t-PA which interact with a panel of twenty-three monoclonal anti-t-PA antibodies. Twenty were directed against domains on the heavy chain, two against the "finger" domain, three against the "epidermal growth factor-like" domain, five against the kringle 1 domain, and ten against the kringle 2 domain. Only three monoclonal anti-t-PA antibodies interact with the light chain. The finding that the epitopes of each of the monoclonals could be determined with the deletion mutant proteins supports the hypothesis of autonomous folding of structural domains and emphasizes the validity of the use of the recombinant t-PA-deletion mutant proteins for structure-function studies.

Antibodies, Monoclonal↗
PubMed1987

Expression of variant von Willebrand factor (vWF) cDNA in heterologous cells: requirement of the pro-polypeptide in vWF multimer formation.

Von Willebrand factor (vWF) is a multimeric plasma glycoprotein synthesized by vascular endothelial cells as a pre-pro-polypeptide with a highly repetitive domain structure, symbolized by the formula: (H)-D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-(OH) A heterologous expression system for the synthesis of recombinant vWF protein was developed, consisting of a monkey kidney cell line (COS-1), transfected with full-length vWF cDNA. This system was shown to mimic the constitutive secretory pathway of vWF in endothelial cells, since dimerization and multimerization occur similarly. To determine whether the pro-polypeptide, composed of the domains D1 and D2, is involved in vWF multimerization, a vWF cDNA was constructed that lacked the coding sequence for the pro-polypeptide. The mutant vWF protein, expressed by COS-1 cells transfected with this cDNA, did not assemble beyond the dimer stage. From this observation, we conclude that (i) dimerization does not involve the pro-polypeptide of pro-vWF and (ii) the presence of the pro-polypeptide, as part of pro-vWF, is obligatory for multimerization. It is argued that the interactions, required for interchain binding, are mediated by the D domains.

Animals↗
PubMed1986

On the interaction of the finger and the kringle-2 domain of tissue-type plasminogen activator with fibrin. Inhibition of kringle-2 binding to fibrin by epsilon-amino caproic acid.

The binding of tissue-type plasminogen activator (t-PA) to fibrin is mediated both by its finger domain and by its kringle-2 domain. In this report, we investigate the relative affinities of these domains for lysine. Human recombinant t-PA deletion-mutant proteins were prepared and their ability to bind to lysine-Sepharose was investigated. Mutants containing the kringle-2 domain bound to lysine-Sepharose, whereas mutants lacking this domain but containing the finger domain, the epidermal growth factor domain or the kringle-1 domain did not bind to lysine-Sepharose. Mutant proteins containing the kringle-2 domain could be specifically eluted from lysine-Sepharose with epsilon-amino caproic acid. This lysine derivative also abolished fibrin binding by the kringle-2 domain but had no effect on the fibrin-binding property of the finger domain. Thus, a lysine-binding site is involved in the interaction of the kringle-2 domain with fibrin but not in the interaction of the finger domain with fibrin. The implications of the nature of these two distinct interactions of t-PA with fibrin on plasminogen activation by t-PA will be discussed.

Aminocaproates↗
PubMed1986

Quantification of tissue-type plasminogen activator (t-PA) mRNA in human endothelial-cell cultures by hybridization with a t-PA cDNA probe.

We describe the construction of a recombinant DNA plasmid, consisting of the vector pBR322 and full-length tissue-type plasminogen-activator (t-PA) cDNA, by using polyadenylated RNA from cultured Bowes melanoma cells as substrate. A 1280-base-pair PstI restriction fragment, covering the 3' untranslated region and part of the coding region for the t-PA L-chain, was used as a radiolabelled probe to determine the size and the number of t-PA mRNA molecules in cultured endothelial cells of different origin from the same individual. Northern blotting showed that in all these cells a t-PA mRNA is synthesized of about 2500 nucleotides, indicating that transcriptional initiation, splicing and polyadenylation is similar. The number of t-PA mRNA molecules per cell measured, by using a dot-blotting technique and t-PA mRNA made in vitro, with a plasmid DNA preparation harbouring a specific promotor of the Salmonella typhimurium bacteriophage SP6, t-PA cDNA and SP6 RNA polymerase as standard, is approx. 10,000 in all cultured endothelial cells from adult vessels. However, the amount of t-PA antigen synthesized and/or secreted differs by a factor of 6-20. Relatively large amounts of t-PA antigen secreted were detected in conditioned medium from vena-cava-derived cells, whereas low amounts were found in conditioned medium from arteria-iliaca-derived cells.

Antigens↗
PubMed1986

Full-length von Willebrand factor (vWF) cDNA encodes a highly repetitive protein considerably larger than the mature vWF subunit.

Full-length human von Willebrand factor (vWF) cDNA was assembled from partial, overlapping vWF cDNAs. This cDNA construct includes a coding sequence of 8439 nucleotides which encode a single-chain precursor of 2813 amino-acid residues, representing a putative signal peptide, a prosequence and mature vWF of 22, 741 and 2050 amino acids, respectively. This represents the longest coding sequence determined to date. In-vitro expression of full-length vWF cDNA revealed the synthesis of a polypeptide with a mol. wt corresponding with that of the unglycosylated precursor. The precursor is a highly repetitive protein which consists of two duplicated (B, C), a triplicated (A), a quadruplicated (D) and a partly duplicated domain (D'), in the following order: H-D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-C1-C2-OH. Both the prosequence, composed of two D domains (D1, D2), and mature vWF harbor an arg-gly-asp ('R-G-D') sequence which has been implicated in cell-attachment functions. It is argued that the pro-sequence is equivalent to von Willebrand Antigen II (vW AgII).

Amino Acid Sequence↗
PubMed1986

Endothelial plasminogen activator inhibitor (PAI): a new member of the Serpin gene family.

A human endothelial cDNA expression library, based on the Escherichia coli plasmid pUC9, was screened with a heterologous antibody raised against purified bovine aortic endothelial plasminogen activator inhibitor (PAI). A synthetic oligonucleotide, derived from a partial PAI cDNA expression clone, was used to select a full-length PAI cDNA, the size of which coincides with the length of PAI mRNA (approximately 2350 nucleotides) as determined by Northern blot analysis. The authenticity of full-length PAI cDNA is demonstrated by the expression of biologically active PAI both in lysates of transformed E. coli cells and in conditioned media of mouse Ltk- cells, transfected with PAI cDNA inserted into vector pSV2. Analysis of the de novo synthesized anti-plasminogen activator activity, employing reverse fibrin autography, shows that transfected mouse Ltk- cells synthesize a polypeptide with a mol. wt identical to that of the native PAI glycoprotein (Mr 52,000), whereas in E. coli an unglycosylated, active product with a mol. wt of 43,000 is made. The amino acid sequence, derived from the determined nucleotide sequence, shows that pre-PAI consists of 402 amino acids. It is proposed that the mature PAI is preceded by a signal peptide of 23 amino acid residues. The amino acid sequence of mature PAI includes three potential asparagine-linked glycosylation sites and lacks cysteine residues. The predicted amino acid sequence reveals significant homology with members of the serine protease inhibitor (Serpin) family, e.g. alpha 1-proteinase inhibitor and antithrombin III.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1986

Structure and function of human tissue-type plasminogen activator (t-PA).

Full-length tissue-type plasminogen activator (t-PA) cDNA served to construct deletion mutants within the N-terminal "heavy" (H)-chain of the t-PA molecule. The H-chain cDNA consists of an array of structural domains homologous to domains present on other plasma proteins ("finger," "epidermal growth factor," "kringles"). These structural domains have been located on an exon or a set of exons. The endpoints of the deletions nearly coincide with exon-intron junctions of the chromosomal t-PA gene. Recombinant t-PA deletion mutant proteins were obtained after transient expression in mouse Ltk- cells, transfected with SV40-pBR322-derived t-PA cDNA plasmids. It is demonstrated that the serine protease moiety of t-PA and its substrate specificity for plasminogen is entirely contained within the C-terminal "light" (L)-chain of the protein. The presence of cDNA, encoding the t-PA signal peptide preceding the remaining portion of t-PA, suffices to achieve secretion of (mutant) t-PA into the medium. The stimulatory effect of fibrin on the plasminogen activator activity of t-PA was shown to be mediated by the kringle K2 domain and, to a lesser extent, by the finger domain. The other domains on the H-chain, kringle K1, and the epidermal growth-factor-like domain, do not contribute to this property of t-PA. These findings correlate well with the fibrin-binding properties of the rt-PA deletion-mutant proteins, indicating that stimulation of the activity is based on aligning of the substrate plasminogen and its enzyme t-PA on the fibrin matrix. The primary target for endothelial plasminogen activator inhibitor (PAI) is located within the L-chain of t-PA. Deleting specific segments of t-PA H-chain cDNA and subsequent transient expression in mouse Ltk- cells of t-PA deletion-mutant proteins did not affect the formation of a stable complex between mutant t-PA and PAI.

Amino Acid Sequence↗