Calibration of the Ph. Eur. BRP Batch 3/Mega 2 (US/FDA) standard for human coagulation factor VIII concentrate for use in the potency assay.

The European Pharmacopoeia Biological Reference Preparation Batch 3/Mega 2 (United States/Food and Drug Administration) (Ph. Eur. BRP Batch 3/Mega 2 (US/FDA)) was developed as an internationally available, common working standard to replace the dwindling stocks of Mega 1 (the current US standard) and Ph. Eur. BRP Batch 2 (the current European standard). The potency was assigned in an international collaborative study with reference to four currently established standards, Ph. Eur. BRP batch 2, WHO 5th and 6th International Standard and Mega 1. Thirty-eight laboratories participated in the collaborative study. Each laboratory was asked to perform four independent assays. Participants used either the one stage clotting assay or the chromogenic assay or both. This publication reports the results obtained with both assays. The summary and conclusion, however highlight the results mainly with respect to the chromogenic assay, which is the assay prescribed in the European Pharmacopoeia. Data were analysed for both assays separately. A consensus potency value was calculated as the unweighted average of mean potencies determined against the four standards. A potency of 8.6 IU/vial as determined in the chromogenic substrate method was assigned to the candidate standard. Inter-laboratory agreement as assessed by calculation of the geometric coefficient of variation was below 10% for mean potencies against all four calibrators for the chromogenic assay. Ph. Eur. BRP Batch 3/Mega 2 (US/FDA) is a freeze-dried, plasma derived, high-purity concentrate. The material was filled into approximately 100,000 vials and lyophilised to a final residual moisture of < or = 2%. Approximately 90,000 vials of the standard are available, equally shared between the two co-ordinating centers. Based on the stability studies, the predicted mean percentage loss per year at -20 degrees C is 0.000% and thus the candidate standard appears to be stable. The Ph. Eur. BRP batch 3 was adopted by the European Pharmacopoeia Commission in November 2001.

Inhibition of human umbilical vein endothelial cell proliferation by the CXC chemokine, platelet factor 4 (PF4), is associated with impaired downregulation of p21(Cip1/WAF1).

Human PF4 is a heparin-binding chemokine known to be capable of inhibiting endothelial cell proliferation and angiogenesis. To explore the biological mechanisms responsible for this action, we investigated the effect of PF4 on epidermal growth factor (EGF)-stimulated human umbilical vein endothelial cells (HUVEC), a model system in which stimulation is essentially independent of interaction with cell-surface glycosaminoglycans. Based on previous findings that PF4 blocks endothelial cell cycle entry and progression into S phase, we studied the molecular mechanism(s) of PF4 interference with cell cycle machinery. PF4 treatment of EGF-stimulated HUVEC caused a decrease in cyclin E-cyclin-dependent kinase 2 (cdk2) activity with resulting attenuation of retinoblastoma protein phosphorylation. PF4-dependent downregulation of cyclin E-cdk2 activity was associated with increased binding of the cyclin-dependent kinase inhibitor, p21(Cip1/WAF1), to the cyclin E-cdk2 complex. Analysis of total cellular p21(Cip1/WAF1) showed that in the presence of PF4, p21(Cip1/WAF1) levels were sustained at time points when p21(Cip1/WAF1) was no longer detectable in cells stimulated by EGF in the absence of PF4. These findings indicate that PF4 inhibition of HUVEC proliferation in response to EGF is associated with impaired downregulation of p21(Cip1/WAF1) and provide the first evidence for interference with cell cycle mechanisms by a chemokine.

Postmortem DNA diagnosis of factor V Leiden in a neonate with systemic thrombosis and probable antithrombin deficiency.

BACKGROUND: Spontaneous neonatal thrombosis due to heritable gene defects has been reported in the past. A recently discovered defect, the factor V Leiden mutation, is the most frequent inherited risk factor for venous thrombosis. CASE: Factor V Leiden was diagnosed postmortem in a neonate who died from complications of vena caval and aortic thrombosis. Investigation into the family history revealed that the father had a record of multiple thromboses, and blood testing demonstrated that the father had antithrombin deficiency and the mother was heterozygous for factor V Leiden. Although we were unable to demonstrate directly the presence of antithrombin deficiency in the infant, we propose that a combination of the two inherited disorders was likely the cause of fatal neonatal thrombosis. CONCLUSION: The present report highlights the importance of a complete prenatal genetic analysis, including factor V Leiden testing and antithrombin measurement in families with a history of thrombotic disorders.

Characterization of the human platelet/endothelial cell adhesion molecule-1 promoter: identification of a GATA-2 binding element required for optimal transcriptional activity.

Platelet/endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kD member of the Ig gene superfamily that is expressed on platelets, endothelial cells, and certain leukocyte subsets. To examine the factors controlling vascular-specific expression of PECAM-1, we cloned the 5'-flanking region of the PECAM-1 gene and analyzed its transcriptional activity. 5'-Rapid amplification of cDNA ends (5'-RACE) analysis showed that transcription initiation occurred at several closely spaced nearby sites originating approximately 204 bp upstream from the translation start site. Analysis of the sequence immediately upstream from the transcription initiation site (TIS) showed no canonical TATA or CAAT elements, however an initiator element commonly found in TATA-less promoters encompassed the TIS. 5'-serially truncated PECAM-1 promoter segments cloned in front of a luciferase reporter drove transcription in both a lineage- and orientation-specific manner. Putative cis-acting control elements present within a 300-bp core promoter included two ets sites, an Sp1 site, tandem E-box domains, two GATA-associated sites (CACCC), an AP-2 binding site, and a GATA element at -24. Mutational analysis showed that optimal transcriptional activity required the GATA sequence at position -24, and gel-shift assays further showed that the GATA-2 transcription factor, but not GATA-1, bound to this region of the PECAM-1 promoter. Understanding the cis- and transacting factors that regulate the tissue-specific expression of PECAM-1 should increase our understanding of the mechanisms by which vascular-specific gene expression is achieved.

The sensitivity of allele-specific polymerase chain reaction can obviate concern of maternal contamination when fetal samples are genotyped for immune cytopenic disorders.

OBJECTIVE: Fetuses at risk for immune cytopenic disorders can be identified by molecular genotyping assays. To better understand the impact of maternal contamination on genotyping results, the levels of contamination that are routinely encountered during prenatal testing of fetal samples and the sensitivity of allele-specific polymerase chain reaction in detecting paternal alloalleles were examined. STUDY DESIGN: Reconstitution experiments were performed to define the sensitivity of allele-specific polymerase chain reaction assays. The sensitivities of allele-specific polymerase chain reactions and polymerase chain reaction-restriction fragment length polymorphism were compared for detection of the factor V Leiden mutation. RESULTS: A quantitative analysis of variable-number tandem repeat loci revealed maternal contamination in 4 of 56 fetal samples. Contaminating deoxyribonucleic acid compromised genotyping results when it comprised between 94% and 99% of the total deoxyribonucleic acid. Allele-specific polymerase chain reaction was found to be the more sensitive technique (0.8% sensitivity vs 13% sensitivity). CONCLUSION: These results illustrate that allele-specific polymerase chain reaction is well suited for reliable prenatal identification of fetuses at risk of immune cytopenic disorders.

The human PECAM1 gene maps to 17q23.

We have determined the chromosomal and regional location of the gene encoding PECAM-1 (termed PECAM1 by GBD nomenclature) using a polymerase chain reaction (PCR)-based analysis of somatic cell hybrids. Analysis of a somatic cell hybrid chromosome panel established that the PECAM1 gene is on chromosome 17. Interestingly, several adhesion molecules expressed on platelets and endothelium also localize to chromosome 17: the GP1BA locus (glycoprotein (GP) Ibalpha) has been provisionally mapped to the region 17p12-pter, the ITGA2B (GPIIb) and the ITGB3 (GPIIIa) loci have been confirmed to the region 17q21.32; and the ICAM2 locus has been provisionally mapped to the region 17q23-q25. To determine if the PECAM1 locus colocalizes with any of the loci for these adhesion molecules, PCR-based analysis of a regional mapping panel for human chromosome 17 was conducted. We found that the PECAM1 locus is on the long arm of chromosome 17, in the region q23-qter. To confirm this observation and obtain a more precise localization of the PECAM1 locus, fluorescence in situ hybridization was conducted. Together our data allowed assignment of the PECAM1 locus to the region 17q23.

Activated protein C resistance in a neonate with venous thrombosis.

A term infant had a life-threatening inferior venal caval thrombosis during the first 24 hours of life. The plasma from the infant and his mother was found to be resistant to activated protein C and to be heterozygous for the factor V mutation (FV Leiden) associated with this disorder. The presence of this hereditary disorder should be considered in infants with thrombosis and in infants with conditions predisposing them to thrombosis.

The polymerase chain reaction with sequence specific primers for the detection of the factor V mutation associated with activated protein C resistance.

The prevalence of the Factor V (FV) mutation associated with activated protein C resistance (FV Leiden) and its significance as a genetic risk factor for venous thrombosis have necessitated the development of a simple, rapid, and accurate assay for its detection. The polymerase chain reaction with sequence specific primers (PCR-SSP) provides a powerful technique for the discrimination of alleles resulting from single base substitutions. PCR amplification was performed using a sense primer complementary to both FV alleles coupled with either of two antisense allele specific primers, one complementary to the normal FV allele and one complementary to the FV Leiden allele. PCR conditions were developed that favored amplification only in the case of perfect complementation between template DNA and allele specific primer. The FV genotype was assigned based on whether or not each allele specific primer set produced an amplified product. Assignment of genotypes correlated 100% with those determined by the method of PCR amplification followed by Mn1I digestion. PCR-SSP allows the rapid and accurate identification of carriers of the Factor V Leiden mutation by a simple PCR reaction without the need for the usual post-amplification specificity step.

Organization of the gene for human platelet/endothelial cell adhesion molecule-1 shows alternatively spliced isoforms and a functionally complex cytoplasmic domain.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a cell-cell adhesion molecule that is expressed on circulating platelets, on leukocytes, and at the intercellular junctions of vascular endothelial cells and mediates the interactions of these cells during the process of transendothelial cell migration. The cDNA for PECAM-1 encodes an open reading frame of 738 amino acids (aa) that is organized into a 27-aa signal peptide, a 574-aa extracellular domain composed of 6 Ig homology units, and a relatively long cytoplasmic tail of 118 aa containing multiple sites for posttranslational modification and postreceptor signal transduction. To provide a molecular basis for the precise evaluation of the structure and function of this transmembrane glycoprotein, we have determined the organization of the human PECAM-1 gene. The PECAM-1 gene, which has been localized to human chromosome 17, is a single-copy gene of approximately 65 kb in length and is broken into 16 exons by introns ranging in size from 86 to greater than 12,000 bp in length. Typical of other members of the Ig superfamily, each of the extracellular Ig homology domains is encoded by a separate exon, consistent with PECAM-1 having arisen by gene duplication and exon shuffling of ancestral Ig superfamily genes. However, the cytoplasmic domain was found to be surprisingly complex, being encoded by seven short exons that may represent discrete functional entities. Alternative splicing of the cytoplasmic tail appears to generate multiple PECAM-1 isoforms that may regulate phosphorylation, cytoskeletal association, and affinity modulation of the mature protein. Finally, a processed pseudogene having 76% identity with PECAM-1 cDNA was identified and localized to human chromosome 3. These findings should have important implications for structure/function analysis of PECAM-1 and its role in vascular adhesive interactions.

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31): alternatively spliced, functionally distinct isoforms expressed during mammalian cardiovascular development.

The establishment of the cardiovascular system represents an early, critical event essential for normal embryonic development. An important component of vascular ontogeny is the differentiation and development of the endothelial and endocardial cell populations. This involves, at least in part, the expression and function of specific cell surface receptors required to mediate cell-cell and cell-matrix adhesion. Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) may well serve such a function. It is a member of the immunoglobulin superfamily expressed by the entire vascular endothelium in the adult. It is capable of mediating adhesion by a heterophilic mechanism requiring glycosaminoglycans, as well as by a homophilic, glycosaminoglycan independent, mechanism. It has been shown to regulate the expression of other adhesion molecules on naive T cells. This report documents by RT-PCR and immunohistochemical analysis the expression of PECAM-1 during early post implantation mouse embryo development. PECAM-1 was expressed by early endothelial precursors first within the yolk sac and subsequently within the embryo itself. Interestingly, embryonic PECAM-1 was expressed as multiple isoforms in which one or more clusters of polypeptides were missing from the cytoplasmic domain. The sequence and location of the deleted polypeptides corresponded to exons found in the human PECAM-1 gene. The alternatively spliced isoforms were capable of mediating cell-cell adhesion when transfected into L-cells. The isoforms differed, however, in their sensitivity to a panel of anti-PECAM-1 monoclonal antibodies. These data suggest that changes in the cytoplasmic domain of PECAM-1 may affect its function during cardiovascular development, and are consistent with our earlier report that systematic truncation of the cytoplasmic domain of human PECAM-1 resulted in changes in its ligand specificity, divalent cation and glycosaminoglycan dependence, as well as its susceptibility to adhesion blocking monoclonal antibodies. This is the first report of naturally occurring alternatively spliced forms of PECAM-1 having possible functional implications.

Characterization of adhesion of "resting" and stimulated platelets to fibrinogen and its fragments.

Adhesion of resting and stimulated platelets to immobilized fibrinogen (Fg) was characterized using various forms of Fg, receptor peptide mimics, and antibodies to glycoprotein (GP) IIb/IIIa and Fg. Resting platelets adhered to Fg, but to less than half the extent of the same platelets stimulated with epinephrine/ADP. The adhesion of resting and stimulated platelets to Fg was inhibited by a receptor peptide mimic (G13, a peptide corresponding to residues 300-312 of GPIIb), anti-GPIIb/IIIa antibodies, and a monoclonal antibody (4A5) against the carboxyl terminus of the gamma chain of Fg. The results presented here demonstrate that the alpha chain RGD platelet recognition sites are not required to mediate the adhesion of either stimulated or resting platelets to immobilized Fg. Although stimulated platelets can adhere extensively to monomeric Fg containing one functional gamma chain, resting platelets require bivalent Fg containing two functional gamma chains to mediate irreversible adhesion to Fg.

Characterization of the gamma chain platelet binding site on fibrinogen fragment D.

Glycoprotein (GP) IIb/IIIa on adenosine diphosphate (ADP)-activated human platelets interacts with specific sites on the fibrinogen molecule leading to aggregation. We characterized the platelet-binding site on the gamma chains of fibrinogen using plasmic fragments D gamma A and D gamma'. Fragment D gamma A, which contains the carboxy terminal gamma A400-411 platelet-binding sequence (HHLGGAKQAGDV), was 70-fold more active than the synthetic gamma A400-411 peptide in inhibiting ADP-induced platelet aggregation. Fragment D gamma A inhibited fibrinogen binding and also bound directly to ADP-activated platelets. The Kd values determined for fibrinogen and fragment D gamma A binding were 0.55 mumol/L and 1.2 mumol/L, respectively. In contrast, fragment D gamma', which differs from fragment D gamma A with respect to its gamma chain sequence from position 408 to the COOH-terminus at position 427, did not inhibit platelet aggregation or fibrinogen binding, and did not bind directly to the platelet surface. Denaturation of fragment D gamma A with guanidine-HCl caused a loss of inhibitory activity in platelet aggregation assays. These data indicate that the native conformation of the gamma chain platelet-binding site on fibrinogen is important for optimal binding to GPIIb/IIIa.

Pantothenate kinase activity in livers of genetically diabetic mice (db/db) and hormonally treated cultured rat hepatocytes.

Preparations from livers of fed and fasted genetically diabetic and nondiabetic mice (C57BL/KsJ db/db, db/+, or +/+) were used to determine whether changes in pantothenate kinase activity and/or properties corresponded to hormonally directed changes in liver total CoA content. Livers of fasted, nondiabetic mice had ratios of pantothenate kinase (PAK) to lactate dehydrogenase (LDH) activity 1.6 times values for fed nondiabetic controls, and they had a total CoA content per milligram of DNA that was 1.8 times control values. Livers of fed genetically diabetic mice had values for PAK/LDH and total CoA per milligram of DNA that were 1.5 and 2.8 times, respectively, those of nondiabetic controls. Liver PAK from genetically diabetic mice was inhibited by acetyl-CoA to the same extent as enzyme from nondiabetic mice and by CoASH to nearly the same extent. Rat hepatocytes in primary culture incubated with dibutyryl cAMP + theophylline + dexamethasone had PAK/LDH levels 1.5 times those of cells not treated with hormonal effectors, and PAK was inhibited to the same extent by acetyl-CoA and nearly the same extent by CoASH. The data show an increase in extractable hepatic PAK activity under conditions in which the total CoA content is elevated, and they suggest that glucocorticoids and cAMP levels contribute to the increased PAK activity.

A unique proteolytic fragment of human fibrinogen containing the A alpha COOH-terminal domain of the native molecule.

The COOH-terminal portion of the A alpha chain of human fibrinogen is highly susceptible to proteolytic degradation. This property has prevented isolation of the COOH-terminal domain of fibrinogen for the direct investigation of its functional characteristics. Human fibrinogen was degraded with hementin, a fibrinogen-olytic protease from the posterior salivary glands of the leech, Haementeria ghilianii. Two initial fragments, Yhem1 and Dhem1, produced by cleavage through the three polypeptide chains in the connector region, were characterized and shown to retain the entire A alpha COOH-terminal domain. Late cleavages by hementin occurred in the A alpha chain COOH-terminal region to produce fragments Yhem and Dhem with shorter A alpha chain remnants. Fragments Dhem were isolated from an intermediate hementin digest of fibrinogen using anion-exchange chromatography. Fragment Dhem1 was separated further from Dhem fragments with shorter alpha chain remnants by affinity chromatography on immobilized plasma fibronectin. Fragment Dhem1 represents a unique proteolytic fragment of fibrinogen containing an intact A alpha chain COOH-terminal region. NH2-terminal sequence analysis of isolated chains from fragment Dhem1 located hementin cleavage sites in the connector region to A alpha Asn102-Asn103, B beta Lys130-Gln131, and gamma Pro76-Asn77. The specific interaction of fragment Dhem1 with immobilized fibronectin indicated that the binding site probably was located within the COOH-terminal 111 amino acids of the A alpha chain. The overall pattern of fibrinogen cleavage by hementin is similar to that of plasmin, yet hementin cleaves preferably in the coiled-coil connector, sparing the A alpha COOH-terminal domain.