bluuebunny/crossref_metadata_2025 huggingface dataset:
ENDOTHELIUM-INDEPENDENT PARTIAL AGONISTIC ACTIVITY OF VASOPRESSIN IN THE RAT COMMON CAROTID ARTERY
Authors: Katušić Z S , Krstić M K
Published: August 2018

It has been shown that vasopressin causes endothelium-dependent relaxations of the canine basilar artery, suggesting that_increased circulating concentrations of vasopressin can produce redistribution of the blood from the peripheral to the cerebral circulation (Katušić et al., 1984). The present experiments were done to examine effect of vasopressin on the rat peripheral arteries* The experiments were performed on rings (4 mm long) of aorta, common carotid and renal arteries* The arteries were placed in Krebs-Ringer-bicarbonate solution gassed with 95%O2-5%co2 gas mixture and kept at 37°c. In certain rings the endothelium was removed mechanically by gentle rubbing of the intimal surface. Isometric tension was continously recorded (IPM electronic).Vasopressin (10-11 to 3×10-8 M) caused concentration-dependent contraction being full agonist in aorta and renal artery, but partial agonist in common carotid artery. After removal of endothelium vasopressin-induced contractions were not significantlly afected in any of the tested arteries. Gyclooxygenase inhibitor indomethacin (10-5 M; contact time = 40 min) did not affect partial agonistic activity of vasopressin in common carotid artery. This results indicate that partial agonistic activity of vasopressin in common carotid artery is not related to increased production of "EDRF" or prostacyclin from endothelium. In addition, our findings are consistent with the hypothesis that vasopressin may contribute to redistribution of the blood flow from the peripheral to the cerbral circulation.

Z.S. Katušić, J.T. Shepherd and P.M. Vanhoutte. Circ. Res., 55: 575-579, 1984
ENDOTHELIAL CELL INJURIES AND SMOOTH MUSCLE CELL PROLIFERATION INDUCED BY MATERIALS RELEASED FROM PLATELET-RICH THROMBUS IN VIVO
Authors: Asada Y , Hayashi T , Sumiyoshi A
Published: August 2018

It is widely held that the disturbance in the integrity of the arterial endothelium may lead to the development of arteriosclerosis and many factors have been postulated to cause the endothelial injury, such as hemodynamic stress, anoxia, platelet-releasing materials, and so on. However, whether any of these is important for endothelial injury is unclear. We studied whether the released products from activated platelets and/or thrombi could cause endothelial damage and proliferation of smooth muscle cells in large vessels in vivo.

Polyethylene tubing was inserted into the ascending aorta of rabbits via the common carotid artery and placed for one, 4, and 24 weeks continuously to induce vessel wall injury and thrombotic events. Then the direct non-injured segments from the descending thoracic and abdominal aorta was morphologically examined, and 3H-thymidine incorporation into the arterial wall was also examined. The descending aortas of experimental rabbits showed endothelial damage and increased mitoses of endothelial cells. Modified smooth muscle cells were noted in the subendothelial layer, and 3H-thymidine incorporation into the intima and media significantly increased in the experimental group at one week(Fig.). At 24 weeks, the intimal thickening with smooth muscle cell proliferation was also found.

This experiment indicates that materials released from the activated platelets and/or thrombi into the circulation can cause endothelial damage and smooth muscle cell proliferation and intimal thickening at downstream and remote aortic segments.
MODULATORY EFFECT OF SERINE PROTEASES AND RELATED ENZYMES ON ISOLATED SMOOTH MUSCLE PREPARATIONS
Authors: Kindel G , Fareed J
Published: August 2018

Thrombin and related proteases produce varying pharmacologic responses in animal models. To more specifically study the in vivo actions of thrombin and related proteases, we have used isolated tissue preparations of the rabbit aortic strip (RAS), isolated guinea pig ileum (GPI) and isolated rat uterus (RU). Standard tissue-agonist regimens include epinephrine, thromboxane B2 with RAS; bradykinin, acetylcholine, histamine and serotonin with GPI; and acetylcholine, bradykinin and angiotensin with RU. The smooth muscle modulant action of numerous proteinases were screened in these regimens by bracketing the median dose response of the individual agonists. Protease complexes such as serum (rabbit, human and guinea pig), activated and nonactivated prothrombin complex concentrates and pancreatin were shown to produce varying but similar contractile responses as obtained by the standard agonists. Sera produced a dose-dependent contraction of the RAS, GPI and RU preparations. Various forms of thrombin produced different degrees of contraction of RAS accompanied by a desensitization process. On a molar basis the order of contractile activity ranged α > β>γ > nitro > DIP. All thrombins were found to augment the epinephrine and thromboxane B2 induced contraction of the RAS. Bovine and human factor Xa produced marked dilatation of the RAS but did not have any effect on the GPI and RU preparations. These results suggest that proteases exert direct musculotropic actions on smooth muscles. This should be taken into consideration in the pathophysiology of vascular spasms.
LABORATORY EVALUATION OF A MULTICHANNEL VERTICAL PHOTOMETRIC ANALYZER FOR TEE TESTING OF CLOT, CHROMOGENIC AND ELISA BASED METHODS FOR HEMOSTATIC SYSTEM
Authors: Fabbrini N , Walenga J M , Hoppensteadt D , Fareed J
Published: August 2018

Due to major developments in the molecular understanding of the hemostatic system, many newer tests to evaluate bleeding and clotting disorders have been developed. These newer tests are based on clotting, enzymatic and immunologic techniques. Since routine coagulation laboratories are not equipped with instruments capable of enzymatic or immunologic measurements, adaptation of these newer tests to the clinical laboratory has been rather limited. As these tests have a significant impact on the basic diagnostics of hemostatic disorders and the monitoring of antithrombotic therapy, the need for instruments capable of performing each of these tests is evident. We have evaluated a new multiprobe instrument, the FP-910 Coagulation Analyzer (Lab-Systems, Helsinki, Finland), for numerous clot-based, immunologic and amidolytic assays. This instrument has a programmable microprocessor controlled analyzer, mixer and incubator in a semi-automated system capable of hight throughput due to multiple processing. A unique vertical light path system allows quantification of even particulate reactions. It is compatible with most commercially available reagents for clot-based synthetic substrate and ELISA methodologies. Individual methods are preprogrammed in the instrument and additional methods can be programmed for all detection modes. For the clot-based assays (P.T, APTT, Heptest®) , excellent correlations were obtained with the BBL Fibrometer and General Diagnostics Coagamate X2 (n=50, r>0.95). The chromogenic assay kits from various suppliers (AT III, heparin, plasminogen, a-antiplasmin) also correlar-ted well (n=50, r>0.95). Similarly the ELISA based protein C assay also correlated well with the Dynatech® system (n=50, r>0.98). The instrument is easy to operate, has a high throughput in all modes (50-100/hr.), high reproducibility and is economically feasible for routine laboratories involved in multi-parametric testing of hemostasis.
THE INFLUENCE OF GLUCOCORTICOID HORMONES ON THE GENE TRANSCRIPTION OF POUR COMPONENTS OF THE FIBRINOLYTIC SYSTEM
Authors: Medcalf R L , van den Berg E , Schleuning W-D
Published: August 2018

The hormonal regulation of plasminogen activator (urokinase type (u-PA) and tissue-type (t-PA)) biosynthesis plays an important role in fibrinolysis and extracellular matrix turnover during invasive growth and cell migration. Recently, two genetically distinct inhibitors of both PA's (PA inhibitor 1 (PAI-1) and PA inhibitor 2 (PAI-2)) have been described which may contribute to the modulation of matrix stability. We have employed cloned cDNA probes to study the regulation of biosynthesis of these proteins in the human fibrosarcoma line HT1080. These cells constitutively express high levels of pro-u-PA. PAI-1, PAI-2 and t-PA are also present at relatively low levels. Treatment of the cells with the glucocorticoid dexamethasone (Dex; 10−7 M), almost completely suppresses u-PA gene transcription, as determined by measurement of in vitro elongation of initiated u-PA transcripts in isolated nuclei ("run-on" transcription assay). Concomitantly, Dex also induces PAI-1 and t-PA gene transcription, whereas PAI-2 gene transcription appeared to remain ' unaffected. These changes in transcription rates are also reflected at the level of mRNA: u-PA mRNA is decreased, whereas PAI-1 and t-PA mRNA are simultaneously induced. PAI-2 mRNA is apparently unchanged. These results demonstrate that glucocorticoid hormones reprogramne the expression of components of the fibrinolytic system, and are of possible relevance in the context of inflammatory disease and malignancy.
PHARMACOKINETICS OF GENETICALLY MODIFIED T-PA IN RAT
Authors: Larsen Glann R , Metzger Mark , Blue Yitzak , Henson Kim
Published: August 2018

To better understand the non-saturable mechanism responsible for the rapid systemic clearance of t-pa, previously demonstrated in all animal species tested so far, we have synthesized a genetically altered form of t-pa and analyzed the pharmacokinetic profile and organ distribution of this mutant. The mutant t-pa has been deleted in the fibronectin finger and epidermal growth factor domain, plus genetically modified to prevent N-linked glycosylation from occurring. Mammalian cells secreting either wild-type or mutant t-pa were radiolabeled with 35S-metnionine. The metabolically labeled t-pa was purified to homogeneity from conditioned medium and injected into the tail vein of rats. Multiple plasma samples were taken, post infusion, and analyzed for levels of acid-precipitable radioactivity. All rats were sacrificed fifteen minutes post infusion to determine the distribution of radioactivity in the liver, kidney or lung. The clearance profile of wild-type t-pa (n=8) was biphasi.e. with an initial alpha-phase half-life of 0.8 minutes (r=0.99) for approximately 80% of the protein and a beta-phase half-life of 12.2 minutes (r=0.97). Essentially all of the radioactivity recovered at fifteen minutes post-infusion was found in the liver. The mutant was analyzed (n=3) in identicle fashion and found to have only a single clearance phase with a 15.4 minute half-life (r=-97). Radioactivity determinations performed on organs showed proportionally less protein to be resident in the liver with a substantial increase in radioactivity observed in the kidneys. In conclusion, we have synthesized a genetic variant of t-pa which has a significantly increased half-life in the rat.
BIOSYNTHESIS AND SECRETION OF β-THROMBOGLOBULIN BY A HUMAN MEGAKARYOBLASTIC CELL LINE ( MEG-01 )
Authors: Ogura M , Tanabe N , Hamaguchi M , Hotta T , Saito H
Published: August 2018

β-Thromboglobulin ( βTG ) is a well known platelet specific a-granular protein, but synthesis of βTG by human megakaryocytes has not been fully proved. A human megakaryoblastic cell line ( MEG-01 ) was investigated for the presence of βJG in the culture medium and cell lysates using a specific radioimmunoassay ( RIA ). The concentration of βTG increased with time in the serum-free culture medium as well as in the cell lysates as shown in the following table.

By an indirect immunofluorescent technique using a monospesific rabbit anti serum against human βTG, βTG antigen was detected in MEG-01 cells. Immunoblot analysis of culture medium revealed a single band ( mol wt 8,900 ) that is identical to the band of human plasma βTG. De novo synthesis of βTG was demonstrated by the presence of specific immunoprecipitable radioactivity in the medium after 5 h of labeling of the cells with [35S]-methionine as a 8,900 mol wt protein. These results indicate that human megakaryocytes produce βTG, The production of βTG by MEG-01 cells may be useful for the study of megakaryocyte maturation and differentiation.
SELECTIVE INHIBITION OF HUMAN MEGAKARYOCYTOPOIESIS IN VITRO BY HIGHLY PURIFIED PLATELET FACTOR 4
Authors: Gewirtz A , Xu W Y , Rucinski B , Niewiarowski S
Published: August 2018

Platelet (plt) factor 4 (PF4) is an alpha granule protein which can modulate T lymphocyte function. T cells may help regulate megakaryocytopoiesis. Therefore, we hypothesized that T cell-PF4 interactions might play a role in autoregulating marrow megakaryocyte (MEG) production. To test this idea, we studied MEG colony formation in plasma clot cultures containing human serum derived solely from pit poor normal AB plasma, enriched hematopoietic progenitor cells (HPC), autologous T cells, and exogenous PF4. Highly purified PF4 (single band on SDS gel) was prepared from outdated human pits by a combination of heparin-agarose, Sephacryl G-200, and Sephadex G-50 column chromatography. HPC were prepared by depleting normal light density marrow mononuclear cells of adherent monocytes, and T cells. T cells were further fractionated into helper (Leu 3+) and suppressor (Leu 2+) subtypes by solid phase immunoabsorption ("panning"). MEG colonies were enumerated by indirect immunofluorescence with an anti-human platelet glycoprotein antiserum. HPC(5×105/ml) were co-cultured with Leu 3+, or Leu 2+ T cells at target;T cell ratios of 2:1 (n=3; n=4 respectively) and l:l(n=4; n=4 respectively) in the presence of 2.5 μg/ml PF4. Under these growth conditions, MEG colony formation was unchanged (p>0.5) when compared to colonies formed by HPC in the absence of PF4. When the above experiments were repeated (n=2-3/condition) at a higher PF4 concentration [25 μg/ml], MEG colony formation was markedly (>60%) inhibited. To determine if PF4 directly inhibited MEG or erythroid progenitor cell growth (CFU-Meg; CFU-E) in vitro, HPC were cloned in PF4 (25μg/ml) without added T cells. Mean ± SEM of MEG and CFU-E derived colonies formed without vs. with PF4 was as follows:

These results suggest that: 1) PF4 may be a non-T cell dependent, lineage specific inhibitor of CFU-MEG, and 2) PF4 may play a role in autoregulating human megakaryocytopoiesis.
A MODEL TO EVALUATE BLOOD CELLS BEHAVIOUR TO CELL ACTIVATION: A DIFFERENCE BETWEEN MEN AND WOMEN
Authors: Hansen J B , Wilsgard L , Olsen J O , Østerud B
Published: August 2018

This study was carried out to see what kind of response the blood cells have to a weak stimuli of lipopolysacc-harides (LPS), a substance that in small quantities may be a small part of the blood system. The parameters tested under this condition were: thromboxane A2 (TxA2) (produced in platelets), prostacyclin (PGI2) produced in white cells (mainly monocytes) and induced thromboplastin synthesis in monocytes.

Heparinized blood from 40 men and 40 women was incubated with 2 ng LPS/ml blood for 2 hours. Blood cells were then either spun down to obtain plasma or mononuclear cells were isolated from the incubated blood followed by the quantitation of thromboplastin. In order to get measureable PGI2 production, liposomes of soya lecithin were added to amplify this production in monocytes (see abstract by Østerud et al. "Monocyte stimulation--". The quantitation of TxB2 in the resultant plasma samples- revealed a highly significant difference in production of TxA2 between men and women in this system. In the group of men a value of 13.0 ± 5.9 ng/ml was found compared to 7.6 ± 5.8 for women (p<0.01). The liposomes had no effect on the TxA2 production. In contrast, the PGI2 production in women was higher than in men. By quantitating 6-keto-PG 1α concent rat i on in the plasma samples it was found that women had 148 ± 53 pg/ml whereas men had 105± 5 pg/rnl (p< 0.001). ft low index of TxA2 is supposed to be beneficial and associated with PGI2 low frequence of coronary heart disease. In the present study this value was estimated to be 51 for women and 124 in men.

A weak but not significant higher thromboplastin activity was found in the stimulated monocytes of men as compared to women (91.4 ± 40.8×10−3/106 cells for men and 74.9 45.6×10−3/ 106 cells for women).

It is concluded that blood cell activation in women is less harmful than in men and this may reflect the lower rate of CHD in women as compared to men.
G PROTEIN REGULATORS OF PHOSPHOLIPASE C AND ADENYLATE CYCLASE IN PLATELETS
Authors: Brass L F , Manning D R , Woolkalis M J
Published: August 2018

The hydrolysis of polyphosphoinositides (PI) by phospholipase C during platelet activation produces two key intracellular messengers, inositol triphosphate and diacylglycerol. This process is thought to be regulated by a guanine nucleotide binding protein referred to as Gp. Although the evidence that Gp exists is compelling, to date it has not been isolated. Uncertainty about its identity has been compounded by variations between tissues in the susceptibility of Gp to pertussis toxin and by reconstitution studies which show that pertussis toxin-inhibited PI hydrolysis can be restored by purified Gi, the pertussis toxin-sensitive G protein which inhibits adenylate cyclase. Therefore, it remains unclear whether Gp represents a new G protein or a second role for Gj. When platelets permeabilized with saponin were incubated with pertussis toxin and 32P-NAD, a single 42 kDa protein was 32P-ADP-ribosylated which co-migrated with the purified a subunit of Gi. Preincubating the platelets with an agonist inhibited labeling of this protein by dissociating the G protein into subunits. The extent of inhibition correlated with the number of toxin-sensitive functions caused by the agonist. Labeling was abolished by thrombin, which inhibited cAMP formation and caused toxin-inhibitable PI hydrolysis. Labeling was partially inhibited by vasopressin and platelet activating factor, which caused toxin-inhibitable PI hydrolysis, but had no effect on cAMP formation and by epinephrine, which inhibited cAMP formation, but did not cause PI hydrolysis. Labeling was unaffected by the TxA2 analog U46619, which neither caused toxin-sensitive PI hydrolysis nor inhibited cAMP formation. These observations suggest that the 42 kDa band may contain a subunits from both Gp and Gi and, in fact, 2D electrophoresis resolved the 42 kDa protein band into two proteins with distinct pi. However, those agonists linked functionally only to Gp or only to Gi decreased the labeling of both proteins. Therefore, our data suggest (1) that Gj and Gp are the same protein and (2) that whether a aiven platelet agonist affects adenylate cyclase or phospholipase C or both depends upon factors extrinsic to the G protein.
BINDING SITE OF VITAMIN K-DEPENDENT PROTEIN S ON C4b-BINDING PROTEIN
Authors: Suzuki K , Nishioka J , Kusumoto H , Deyashiki Y
Published: August 2018

Protein S, a cofactor for activated protein C, reversibly complexes with a regulatory complement component C4b-binding protein (C4bp) in plasma. In plasma of patients with congenital protein S deficiency, most protein S exists as a complex with C4bp, which has no cofactor activity. C4bp (Mw 550,000) is composed of approximately seven subunits with Mw 75,000 which are linked by disulfide bonds near the carboxy1-terminus. We report here about the complex formation between protein S and C4bp particularly on the binding site of protein S on C4bp molecule. Protein S and C4bp were purified from human plasma. Seventeen mouse monoclonal antibodies against C4bp were prepared. Chymotrypsin-digested C4bp was separated on gel filtration into a fragment with Mw 160,000 derived from the carboxyl-terminal core of the intact C4bp and fragments with Mw 48,000 from the amino-terminus. The carboxy1-terminal fragment with Mw 160,000 was found to be composed of approximately seven polypeptides with Mw 25,000, which were linked by disulfide bonds.

The experiments using these fragments and the monoclonal antibodies showed that: (1) Protein S bound not only to the intact C4bp, but also to the fragment with Mw 160,000. (2) The fragment with Mw 160,000 inhibited the binding of protein S to C4bp, but the fragment with Mw 48,000 did not. (3) One of the seventeen monoclonal antibodies blocked the inhibition of C4bp on the cofactor activity of protein S. (4) This antibody inhibited C4bp binding to protein S. (5) The antibody bound to the fragment with Mw 160,000. Based on these results, protein S was suggested to lose its cofactor activity for activated protein C by binding to the carboxyl-terminal core of C4bp where seven subunits are linked by disulfide bonds.
RESTRICTION ANALYSIS AND SOUTHERN BLOTTING OF TOTAL HUMAN DNA REVEALS THE EXISTENCE OF MORE THAN ONE GENE HOMOLOGOUS WITH PROTEIN S cDNA
Authors: Ploos van Amstel J K , van der Zanden A L , Reitsma P H , Bertina R M
Published: August 2018

A deficiency in protein S, the cofactor of activated protein C, is associated with an increased risk for the development of venous thrombosis. It is inherited as an autosomal dominant disorder. To improve the detection of heterozygotes in affected families, we have started to search for restriction fragment length polymorphism (RFLP) in the protein S gene. This study revealed the existence of two genes containing sequences homologous to protein S cDNA.

Three non-overlapping fragments of clone pSUL5, which codes for the carboxy-terminal part of protein S and contains the complete 3' untranslated region, were isolated and used as probes in search for RFLP of the protein S gene.

Surprisingly the non-overlapping probes shared more than one hybridizing band. The hybridization took place under stringent assay conditions.

This observation is contradictory to the intron-exon organization of a gene and suggests the existence of two genes, containing sequences homologous with pSUL5. Both genes could be assigned to chromosome 3 by mapping through somatic cell hybrids. Whether two functional protein S genes are present in the human genome remains to be established.
VON WILLEBRAND'S DISEASE TYPE IIB ASSOCIATED TO A COMPLEX THROMBOCYTOPENIC THROMBOCYTOPATHY
Authors: Batlle J , López-Fernández M F , López-Berges C , Sánchez R , Villarón L G , Zimmerman T S
Published: August 2018

A family bleeding disorder characterized by a new association between Type IIB von Willebrand's disease (vWD) and a complex platelet disfunction, with an intermittent thrombocytopenia is described in two patients from the same generation. The mother and a maternal aunt died having severe bleeding diathesis. The platelet abnormalities included: borderline or slightly low platelet count but moderate thrombocytopenia coincedent with the acute bleeding episodes, giant platelet site with a very heterogeneous distribution width, large number of vesicles in platelets by electron-microscopy recalling the "Swiss-Cheese" platelets, abnormal platelet aggregation induced by ADP, collagen, epinephrin and slightly, by thrombin, defective release of 14C-Serotonin, von Willebrand factor (vWF) and platelet factor 4 induced by thrombin or ADP. DDAVP was given to both patient and a partial and transitory correction of bleeding time, thrombocytopenia, presence of platelet aggregates on smear besides a brief appearence of larger multimers of vWF and an increase in all Factor VIII/von Willebrand Factor (FVIII/vWF) properties were seen. Binding of labeled vWF using radiolabelled monoclonal anti-vWF antibody showed an enhanced binding of the patients' vWF, induced by ristocetin, to either normal or patients' platelets. In contrast, the binding of labeled purified normal vWF induced by thrombin to patients' platelets was decreased as compared with the correspondent control. Thus, both patients have platelet disfunctions characteristic for more than one specific platelet disorder. Several associations between platelet and FVIII/vWF abnormalities have been described. This is the first family presenting association of Type IIB vWD and a complex thrombocytopathy. The inherited or acquired (induced by the abnormal IIB vWF platelet interaction) nature of the abnormality is discussed.
TWO VON WILLEBRAND FACTOR (vWF) GENE POLYMORPHISMS SEGREGATE WITH VON WILLEBRAND'S DISEASE (vWD) TYPE IIA: ASSIGNMENT OF THE DEFECTIVE GENE LOCUS IN vWD TYPE IIA
Authors: Verweij C L , Quadt R , Briët E , Pannekoek H
Published: August 2018

Patients with autosomal, dominant von Willebrand's disease (vWD) type IIA display a decreasedristocetin cofactor activity and lack the large and intermediate size von Willebrand factor (vWF) multimers. As yet the cause for this abnormal vWF protein is not known. In this study we determined whether vWD type IIA is due to a mutation in the vWF gene or by a defect in another gene involved, in for example, vWF processing.

Restriction fragment-length polymorphisms (RFLP's), using the enzymes BgIII and XbaI in conjunction with human vWF cDNA probes, have been described. Restriction endonuclease analysis of genomic vWF DNA revealed that these genetic marker are located within the vWF gene. The vWF gene was determined to comprise about 160 kb and harbors at least 20 exons. The RFLP's were applied to study the segregation of alleles associated withvWD type IIA in a comprehensive, affected family. It is demonstrated that both RFLP's are completely linked with the vWD type-IIA trait. From this finding, we conclude that the defect, causing the vWD type IIA, is most likely due to a mutation in the vWF gene and not to a mutation in another gene involved in, for example, post-translational processing of the vWF protein.
ISOLATION AND CHARACTERIZATION OF THE GENES FOR THE a AND b SUBUNITS OF HUMAN COAGULATION FACTOR XIII
Authors: Ichinose A , Bottenus R E , Loeb K R , Davie E W
Published: August 2018

Factor XIII (plasma transglutaminase, fibrin stabilizing factor) is a plasma protein that plays an important role in the final stages of blood coagulation and fibrinolysis. The molecule occurs in blood as a tetramer (a2b2) consisting of two a. subunits and two b subunits. Recently, we have determined the amino acid sequences for both the a. and b subunits of human factor XIII by a combination of cDNA cloning and amino acid sequence analysis. cDNAs coding for the a (3.8 Kb) and b (2.2 Kb) subunits were used for the screening of human genomic DNA libraries. Among 12 × 106 recombinant phage, ∼30 have been shown to contain the sequences for the a subunit and ∼10 have been shown to contain the gene for the b subunit of factor XIII. The clones coding for the a. subunit span ∼90 Kb and have been characterized by restriction mapping. Southern blotting, and DNA sequencing. Both 5’ and 3’ ends of the genomic clones correspond to the 5’ and 3’portions of the cDNA for the a.subunit of factor XIII. The DNA sequence revealed that the activation peptide released ^thrombin (amino acid residues 137), the first putative Ca2+ binding region (around residue 251), the active Site Cys (amino acid residue 314), and the second putative Ca2+ binding region (around residue 473) are encoded by separate exons. Accordingly, the intervening sequences may separate the a subunit into functional and structural domains. The gene organization for the b subunit will also be presented. (Supported by NIH Grant HL 16919.)
BUTYRATE SPECIFICALLY STIMULATES TISSUE-TYPE PLASMINOGEN ACTIVATOR SYNTHESIS IN CULTURED HUMAN ENDOTHELIAL CELLS
Authors: Kooistra T , van den Berg J A , Tüns H A M , Platenburg G , Rijken D , van den Berg E A
Published: August 2018

In a search for compounds that can enhance tissue-type plasminogen activator (t-PA) synthesis in cultured human endothelial cells we found that dibutyryl cyclic AMP (at a concentration of 2 mM) led to a several-fold increase in t-PA production by endothelial cells over a 24 h incubation period. Further researchshowed that this stimulating effectcould be explained by the slow liberation of butyrate, as the effect could be reproduced by addition of free butyrate to the medium, but notby addition of 8-bromo cyclic AMP. With butyrate, an accelerated accumulation of t-PA antigen in the conditioned medium (CM) was observed after a lag period of about 6 h. Increasing amounts of butyrate caused anincreasingly stimulatory effect, reaching a plateau with 5 mM butyrate. The relative increase in t-PAproduction in thempresence of 5 mM butyrate varied among different endothelial cell cultures from 6 to 25-fold in 24 h CM. The butyrate-induced increase in t-PA production wasaccompanied by increased t-PA mRNAlevels.

Analysis of radiolabelled CM andcell extracts by SDS-PAGE and autoradiography indicated that the potent action of butyrate is restricted to a limited group of proteins. We found that theaccumulation of PA-inhibitor activity in CM from butyrate-treated cells increased only moderately.

In our study of the relationshipbetween structure and stimulatory activity we found that butyrate wasby far the most effective inducer oft-PA synthesis. Shortening or lengthening the carbon chain by one carbon atom decreased the stimulatory effect after 24 h of incubation by 50-70%. Further changes in the length of the carbon chain almost completely suppressed the stimulatory activity. Similarly, alterations of the aliphatic chain by introduction of functional groups, a double bond or a branched structure rendered butyrate ineffective in increasing t-PA synthesis. Thus, a straight-chain C4 monocarboxylate structure with a methyl group at one end and a carboxyl moiety at the other seems to be required for the rather specific induction of t-PA synthesis in cultured human endothelialcells.
PERSISTENT DECREASED FIBRINOLYTIC ACTIVITY IN CYCLOSPORIN A (CyA) TREATED RENAL ALLOGRAFT RECIPIENTS
Authors: Cohen H , Mackie I J , Patel R , Neild G , Machin S J
Published: August 2018

CyA administration isssociated with histological evidenceof vascular injury. Since vascularendothelial cell components are known to modify fibrinolytic activity, we have examined fibrinolytic changes in renal allograft recipients onCyA and, in addition, markers of vascular endothelial cell damage: plasma vWFRiCof activity and AT-III activity.Parameters of fibrinolytic activity have been assessed serially in 21 renal allograft recipients for 1 year post-transplantation by study of the following: euglobulin clot lysis time (ELT), plasminogen activator activity (PAA) by fibrin plate assay, tissue plasminogen activator (t-PA) by ELISA and fast-acting t-PA inhibitor (t-PAI) by chromogenic assay.

From 1 month onwards, fibrinolytic activity (ELTs and fibrin plates) is significantly decreased, p<0.002 and p<0.002 respectively, compared to normal controls, and this decrease persists at oneyear post-transplantation. Mean ELTs (mins) and ranges at 1 and 6 months and 1 year are 363 (120-1740), 598 (135-1610) and 538(80-870) respectively. Mean PAA (% of pooled normal plasma) and ranges at these times are 104 (70-154), 79 (48-118) and 83 (65-108) respectively.

In contrast, serial t-PA levels (%of pooled normal plasma) are significantly increased (p<0.02), 428 (86-3686), 297 (81-1130) and 198 ( 44-637 ) at 1 and 6 months and 1 year respectively.

These results suggest that the decreased fibrinolytic activity is due to increased levels of t-PA inhibitor, too high to be surpassed by the amount of t-PA released. vWFRiCof activity and AT III activity aresimilarly increased.

In conclusion, CyA-treated renal allograft recipients show evidence of chronic vascular endothelial damage and a prothromboticstate.
ROLE OF PLATELET GLYCOPROTEINS lb AND llb/llla IN TUMOR CELL INDUCED PLATELET AGGREGATION AND TUMOR CELL ADHESION TO EXTRACELLULAR MATRIX. I. Grossi
Authors: Grossi L , Honn K V , Sloane B F , Thomopson J , Ohannesian D , Kendall A , Newcombe M
Published: August 2018

Platelet glycoproteins are known to play a role in platelet platelet interactions, platelet activation, and platelet adhesion to extracellular matrix (ECM). Monoclonal antibody to human platelet glycoprotein lb (mAblb) and polyclonal antibodies to the llb/llla complex (pAbllb/llla) were used to evaluate the involvement of these glycoproteins in tumor cellinduced platelet aggregation (TCIPA and tumor cell adhesion to the ECM. We have demonstrated that human cervical carcinoma (MS5I7), human colon carcinoma (Clone A), and rat Walker 256 carcinosarcoma (W256) cells induce aggregation of homologous platelets via thrombin generation. MAblb and pAbllb/llla were shown to inhibit TCIPA by MS517, Clone A, and W256 in a dose dependent manner. MAblb was also shown to inhibit platelet thromboxane B2 production in response to tumor cells in a dose dependent manner. Neither mAblb nor pAbllb/llla had any effect on ADP stimulated platelet aggregation. Concentrations of mAblb and pAbllb/llla which produced half maximal inhibition alone were combined resulting in complete inhibition of TCIPA. Preincubation of MS5I7 and W256 with mAblb also resulted in inhibition of TCIPA, while preincubation of Clone A with mAblb did not, suggesting the presence of this glycoprotein on the cell membranes of MS5I7 and W256, but not on Clone A. Immunofluorescence studies confirmed the presence of this glycoprotein on the cell plasma membrane of the MS5I7 and W256, but not on Clone A. Preincubation of MS5I7 and W256 with both mAblb and pAbllb/llla alone or in combination, also resulted in decreased (12S)-12 -hydroxy -5, 8,10, 14 -eicosatetraenoic acid (12-HETE) production, while platelets preincubated with these antibodies had no effect on the concentration of 12-HETE produced. Isolation of platelet membranes and released platelet contentswere tested separately and in combination on platelet adhesion to ECM. Platelet release factors were ineffective, while isolated platelet membrane ghosts enhanced adhesion. Disruption of the platelet cytoskeleton andinhibition of the formation of the llb/llla complex decreased platelet enhanced tumor cell adhesion. These findings suggest a role for these platelet glycoproteins in TCIPA, platelet enhanced tumor cell adhesion to ECM and subsequent tumor metastasis.
EFFECT OF RA-233 (MOPIDAMOLE) ON SURVIVAL IN CARCINOMA OF THE LUNG AND COLON. FINAL REPORT OF VA COOPERATIVE STUDY #188
Authors: Zacharski L R , Moritz T E
Published: August 2018

RA-233 (mopidamole) is a phosphodiesterase inhibitor that has been shown previously to limit progression of malignancy in certain experimental animal models and in a pilot study in humans. RA-233 plus chemotherapy was compared with chemotherapy alone in a five-year double-blind trial involving 719 patients with advanced carcinoma of the lung and of the colon. No difference existed between treatment groups for a variety of demographic, clinical, and laboratory parameters evaluated at entry to the study. There were no instances of unblinding and no patients were lost to followup. Minimum followup was 1 year. Patients ingested RA-233 or placebo for over 85% of their total survival interval and took 66% of the number of pills originally prescribed. RA-233 treatment was associated with a statistically significant prolongation of survival in patients with non-small cell lung cancer limited to one hemithorax, and also with reduction in mean plasma fibrinogen concentration, and with reduction in the incidence of bleeding episodes. RA-233 was not toxic.The favorable effects on survival could not be explained by any factor other than the RA-233 treatment. In other tumor categories tested no differences in survival were observed. These results suggest that RA-233 may be useful in the treatment of non-small cell lung cancer of limited extent (and possibly other tumor types). They also suggest that therapeutic intervention aimed at modified pathways within tumor cells might constitute an innovative investigational approach to the treatment of cancer.
DIRECT STIMULATIONOF CA2+-ACTIVATED HUMAN PLATELET PHOSPHOLIPASE A2 BY DIACYLGLYCEROL
Authors: Deykin D , Karmer R M
Published: August 2018

These studies examined the effect of diacylglycerol on Ca2+-dependent phospholipase A2 from human platelets. Phospholipase A2 was solubilized and partially purified to a stable form in the presence of octylglucoside and its enzymatic activity determined using sonicated arachidonoyl phosphatidylcholine (PC) as substrate. (Kramer RM, et al: BBA 878:394, 1986) Phospholipase A2 activity was increased when dioleoylglycerc_ was incorporated into the substrate arachidonoyl-PC. In the presence of 1 uM (29 mol %) sn-1,2-dioleoylglycerol the enzymatic activity was stimulated 4.1-fold. Exogenously added sn-l-oleoyl-2-acetoylglycerol also enhanced phospholipase A2 activity, producing a maximal stimulation of 1.6-fold at a concentration of 25 uM. Comparative studies conducted with pancreatic, bee-venom and snake venom phospholipase A2 showed that the activity of these extracellular phospholipases towards the arachidonoyl-PC substrate was also increased by diacylglycerol, but the stimulation was less than observed for platelet phospholipase A2. We conclude that in stimulated platelets Ca2+-activated phospholipase A2 may be regulated by newly generated diacylglycerols, not only via protein kinase C-mediated events, but also directly through conformational changes imposed by the diglycerides on cellular membrane phospholipids.
HIGH CONCENTRATIONS OF EXOGENOUS ARACHIDONATE INHIBIT CALCIUM MOBILIZATION IN PLATELETS
Authors: Kowalska M A , Rao A K , Disa J
Published: August 2018

Exogenous arachidonic acid (AA) induces platelet aggregation and secretion which are inhibited at higher AA concentrations.To define the mechanisms for platelet inhibition at high AA,we studied the effect of itS2$odium salt on cytoplasmic ionized calcium concentration [Ca ]i, a key modulator of several platelet responses. In platelets suspended in Hepes buffer containing no albumin, peak aggregation and secretion occurred at 2-5 pM AA with partial inhibition above 10-15 pM AA and complete inhj^ition around 25-50 pM AA. In platelets ^aded with quin2, [Ca+2 ]i rose, in presence of 1 mM external Ca , from basal levels of 70-80 nM to peak of 300-500 nM at 2-5 μM AA; this was followed by inhibition to basal levels at 25-50 μM AA. At these AA concentrations there was no cell lysis. Thromboxane B. production, measured using a radioimmunoassay, was not inhibited even at 25 pM AA. Elevated celjlar levels of cAMP inhibit platelet responses including Ca2+ signals and were therefore measured using a radioimmunoassay. Platelet cAMP levels rose, in the presence of theophylline (7mM), from basal levels of 3.4 pmol/102+ plat to 5.5 at 5 μM AA and to 6.8 pmol/108 plat at 50 pM AA; Ca signals, aggregation, and secretion were inhibited with doubling of cAMP levels. On incubation of platelets with adenylate cyclase inhibitor, 2'5' dideoxgdenosine (DDA, 200 μM, 2 min) there was enhancement of peak [Ca +2]and aggregation noted with 15 pM AA; at 25 μM AA peak [Ca2+ ]i rose from 126 nM to 205 nM and aggregation was restored. Incubation with SQ 22,536 (500 μM, 5min), another adenylate cyclase inhibitor, also attenuated the inhibition by high AA levels. Treatment of platelets with aspirin or_BW 755C, a combined lipooxygenase/cyclooxygenase inhibitor, did not pryent the inhibition by high AA levels of aggregation and Ca responses induced by thromboxane analog, U46619 In conclusion, high AA concentrations inhibit cytoplasmic Ca+2 mobilization in platelets which is related to elevation of platelet cAMP through stimulation of adenylate cyclase. We propose that high AA levels inhibit aggravation and secretion by modulating cytoplasmic levels of Ca2+ and cAMP, two major messenger molecules in platelets.
IN VITRO NEUTROPHIL ACTIVATION BY PLASMA OF STORED PLATELET CONCENTRATES
Authors: Muylle L , Van Brussel C , van Bockstaele D R , Peetermans M E
Published: August 2018

A previous study showed an effect of storage time of platelet concentrates (PLC) on the frequency and severity of reactions to platelet transfusion.As most of the leukocytes were removed from the PLC prior to transfusion, it was suggested that at least part of the reactions were caused by the transfusion of cell products, accumulated during conservation of the PLC.This idea is supported by the finding of high histamine levels in PLC after 5 days storage.In order to test for the presence of substances causing neutrophil activation, samples from 7 PLC were taken at various storage times and incubated for 30 minutes at 37°C with neutrophilic granulocytes obtained from healthy donors. Binding of a monoclonal antibody specific for a neutrophil-associated membrane glycoprotein (gp 170) was analyzed by indirect immunofluorescence. (CLB 13.9,kindly given by P. Tetteroo).It is known that the expression of gp 170 is enhanced by stimulation of the neutrophil.

Fluorescence intensity was quantified by flow cytometry (Ortho cytofluorograf 50-H).Results were expressed as an activation ratio (AR) (ratio of the median fluorescence intensity over that of negative control neutrophils).At day 0 no activation was recorded (AR = 1±0.09,n = 7). In contrast, the supernatant plasma of PLC stored for 7 days caused an elevated AR in 4/7 PLC (AR = 3.20±1.20, n = 4).For comparison the AR of the positive control was 3.66±0.63 (n = 5).In 2 PLC activation was already observed after 1 day storage.The increased expression of gp 170 was not correlated with the histamine level, the lactate dehydrogenase level, the leucocyte or platelet count in the PLC.

These preliminary results indicate the presence of factors causing in vitro activation of neutrophils in the supernatant plasma of some stored PLC.Further investigations are needed to confirm these results, to identify the responsible factor(s) and to study the clinical relevance of this finding.
IN VIVO STUDIES ON RED CELLS AND PLATELETS STORED IN HALFSTRENGTH CITRATE
Authors: Prewse C , Bell K , Griffin B
Published: August 2018

We have previously shown that donation of blood into anticoagulants containing half the normal amount of citrate results in a dramatic improvement in the stability of coagulation factor VIII and has no adverse effect on the in vitro qualities of red cells or platelets during storage. To confirm the viability of stored cellular components we are now performing autologous survival studies in healthy volunteers using radiolabelled cells from red cells and platelets stored for 35 and 5 days respectively. Results to date indicate a 24 hour survival of 80% for red cells stored at a haematocrit of 0.70 for 35 days. Infusion of Ill-In oxine labelled platelets after storage for 5 days in full or half-strength citrate gave recoveries of 40% and survivals of 7 days. These encouraging results suggest use of halfstrength citrate may be a route to increasing factor VIII supply without any additional donor recruitment. Further in vitro studies have also been performed on cellular components and reveal adequate in vitro quality for half-strength citrate blood held at room temperature for 20 hours prior to component preparation.
INDIUM-111 PLATELET SURVIVAL STUDIES ON PLATELET CONCENTRATES (PCVSTORED FOR UP TO 14 DAYS
Authors: Holme S , Heaton W A , Hartman P
Published: August 2018

The effect of extended storage on platelet in vitro and in vivo viability was investigated to determine the reliability and efficacy of Indium-111-oxine radiolabeling of long-term stored platelets. The fitness of various mathematical models for measurement of platelet survival and recovery was also evaluated.

33 CPDA-1 PC were prepared and stored for various periods of time (see below) according to standard blood bank methods. The volume of the PC was adjusted to 65-75 mL to prevent pH fall with extended storage. After storage, samples were taken for in vitro assays and for radiolabeling witn Indium-111-oxine using standardized methods. None of the PC had pH<6.3. Afxer reinfusion of the labeled platelets, samples for measurement of radioactivity were taken at three hours and daily for seven days.

The uptake {%) of Indium-111 and loss of platelets during the labeling showed no correlation with storage time; the means of the 33 studies were 71±8% and 35±6%, respectively. There was no significant correlation between the parameters and recovery or survival which suggests reliable labeling of nonviable platelets.
Excellent fit of the survival data to the multiple hit gamma model was found forpall studies as determined by the coefficient of determination, r 2. The survival curves of platelets stored for one and five days were quite linear as shown by r2 ; with longer storage periods they became more exponential as shown by an increasingly better fit (r2 ) to the log model.

Using the gamma model, both in vivo recovery and survival showed a progressive loss with increasing storage duration. This was less evident with the linear model. The loss of in vivo viability paralleled closely a decrease in the in vitro , parameters of platelet quality (discoid shape, hypotonic shock response, ATP content, and oxygen consumption) during storage.In conclusion, this study indicates that extended storage not only yields platelets with reduced recovery, but also platelets with shortened survival.
COAGUIATION, FIBRINLYSIS AND KALLIKREIN ACTIVATION IN SEVERE INFECTION AND SEPSIS : RELATION TO OUTCOME
Authors: Blamback M , Hesselvik F , Brodin B , Maller R , Gaffney R
Published: August 2018

Fatal multiple organ failure following severe infection may be related to early activation of protease cascade systems. The study aimed to relate changes in the below mentioned components to shock and outcome. Of 53 patients with severe infection, 30 did not develop shock (group I); 12 survived septic shock (groupII); and 11 died from organ failure after septic shock (groupIII). No patient had overt DIC. During the first 3 days after admission, blood was sampled daily for assay of: platelet count, fibrinogen, prothrombin complex, F XII, F VIIIiC, vWF:Ag, F VII, F V, anti thrombin, protein C, plasminogen, antiplasmin, plasminogen activator inhibitor (PAi), X-oligcmers, D-dimers, prekalli-krein, functional kallikrein inhibition (fKl), and fibronectin, by chramogenic substrate and inmunochemical techniques. The Proenzyme functioned, index (PFI) ves calculated combining the results of anti thrombin, plasminogen, antiplasmin, prekallikrein and fKL (Aasen, Acta Chir Scand 1985; 522: 211).

Low (p<.001) initial values for F XII, prothrombin complex, F VII, antithrcmbin, protein C, prekallikrein, and fibronectin were seen in all groups. The shock groups (I-III) had in addition significant decreases in platelet count, antiplasmin, and plasminogen. Fibrinogen, F VIII :C, vWF:Ag, X-oligcmers, and D-dimers were significantly higher than normal in all groups. Shock patients had higher X-oligcmers and D-dimers, but lower fibrinogen than non-shock patients. PAi was within the normal range in survivors (I-II), but was elevated ten-fold and increased progressively over 3 days in the non-survivors. vWF:Ag showed a similar progressive increase in non^survivors; these two variables ware the best early indicators of non-survival. PFI was significantly lower in shock patients (II-III), but did not discern between survivors and non-survivors during days 1-3. The results indicate a marked activation of coagulation in patients with severe infection, with more fibrin formation and fibrinolysis in the shock groups. High vWF:Ag and PAi in non-survivors may indicate nmore endothelial damage, and potentially harmful fibrinolysis inhibition.