bluuebunny/crossref_metadata_2025 huggingface dataset:
STUDIES ON SMALL MOLECULAR WEIGHT ADHESION PROTEINS (SAPs) FROM CONNECTIVE TISSUES
Authors: Wasi S , Alles P , Gauthier D , Bhargava U , Farsi J , Aubin J E , Sodeki J
Published: August 2018

We have identified a family of low molecular weight proteins with cell attachment properties in a variety of soft and mineralised connective tissues (Wong et al., Biochem. J. 232, 119, 1985). For further characterisation of these proteins we extracted porcine bones with 4 M guanidine hydrochloride and purified the proteins on a series of gel filtration columns

The purifed SAPs comprise three bands with Mr -14 000 -17 000. All three proteins bound to heparin-sepahrose in both the presence and absence of 4M urea, and when eluted with 2 M NaCl they retained their cell binding capacity. These proteins promoted the adhesion and spreading of a variety of cell types, including normal fibroblasts, osteoblasts, and epithelial cells, and tumour (osteosarcoma) cells. On Western blotting SAPs did not cross-react with antibodies against fibronectin, laminin or type I collagen; however, they were recognised by a monoclonal antibody to human vitronectin, a polyclonal antibody to bovine vitronectin and polyclonal antibody to human somatomedin B. Dose response experiments indicated that maximum attachment of human gingival fibroblasts occurred in the presence or absence of fetal bovine serum on wells precoated with 2.5 μg/cm2 of SAPs. Attachment of cells to these proteins was partially inhibited by the synthetic pentapeptide Gly-Arg-Gly-Asp-Ser. Utilising the nitrocellulose cell binding assay of Hayman et al (J. Cell. Biol. 95, 20, 1982), the cell attachment to these proteins could be completely inhibited by heparin (100 units/mL) whereas up to 1000 units/mL of heparin had no inhibitory effect on cell attachment to fibronectin and vitronectin. The occurrence of these proteins in a variety of connective tissues and their recognition by different cell types may reflect their general biological role in adhesive mechanisms in both hard and soft connective tissues. Currently, we are investigating the relationship between SAPs and vitronectin, since it is possible that SAPs represent a tissue-processed form of vitronectin or may be novel attachment proteins with regions of homology with vitronectin
A SIMPLE AND CONVENIENT FLUORESCENT ASSAY FOR MONITORING ACTIVATION OF COAGULATION FACTORS IX, X, AND PROTHROMBIN USING p-AMINO-BENZAMIDINE
Authors: Monroe D M , Sherrill G B , Roberts H R
Published: August 2018

Blood coagulation consists of a series of protein-protein-cofactor interactions that involve zymogen coagulation factors being activated to trypsin-like serine proteases, ultimately leading to conversion of prothrombin to thrombin. As described by Evans et al. ((1982) J. Biol. Chea. 257, 3014), the fluorescent compound p-aminobenzamidine (pAB) increases its quantum yield when bound to the active site of thrombin (but not when pAB is reacted with coagulation zymogens). We have devised a simple procedure to monitor activation of purified coagulation factors IX, X, and prothrombin using pAB. Human factor X with 150 μM pAB was activated with purified factor X activating protein from Russell’s Viper venom (RVVX). Fluorescence emission was monitored at 376nm with excitation at 336nm. Portions were removed and activation was measured by clotting assay, by assay using tosyl-Gly-Pro-Arg-p-nitroanilide (tosGlyProArgNA), and by polyacrylamide gel electrophoresis; all of which indicated that factor X activation followed a time course identical to the time course described by the pAB fluorescence increase. Factor IX was activated by factor XIa in the presence of pAB; portions were analyzed for clotting activity. Prothrombin was activated to (meizo)thrombin by Ecarin and activity was measured using tosGly-ProArgNA. In both cases the increase in pAB fluorescence was coincident with the time course described by activity assay. The pAB fluorescence assay has several advantages including: 1) activation can be easily and continuously monitored, 2) the extent of modification can be determined by inspection of the progress curve, and 3) the procedure can be used with a wide range of zymogen concentrations. Additionally, pAB can be used to follow activation of modified (pro)enzymes. For instance, activation of bovine des(γcarboxyglutamic acid)factor X by RVVX was found to be 100-fold slower than activation of unmodified bovine factor X. Our results suggest that following p-aminobenzamidine fluorescence is a useful and convenient procedure for monitoring activation of blood coagulation factors IX, X, and prothrombin.
VITAMIN K-DEPENDENT γ-CARBOXYLATION OF FACTOR IX REQUIRES A RECOGNITION SITE CONTAINED WITHIN THE PROPEPTIDE
Authors: Jorgensen M J , Rabiet MJ , Cantor A B , Furie B , Brown C L , Shoemaker C B , Furie B C
Published: August 2018

The vitamin K-dependent proteins, including Factor IX (FIX), are calcium-binding proteins that undergo vitamin K-dependent post-translational modification to convert amino terminal glutamic aoid residues to Gla residues. Sequence homology among the propeptides of these proteins suggests a role for this region in designating the adjacent glutamic acid-rich domain for γ-carboxylation during intraoellular processing. Mutations vere made in the propeptide (residues -1 to -18) of FIX, and the effects on γ-carboxylation were assessed. The human FIX cDNA coding sequenoe was modified using oligonucleotide-directed site-specific mutagenesis and was expressed in Chinese hamster ovary cells. The extent of γ-carboxylation of secreted FIX was determined by (1) ability to interact with conformation-specific antibodies directed against the Gla-dependent, metal-stabilized, native structure of FIX, and (2) direct Gla analysis of the alkaline hydrolysate. Using the unmodified coding sequence, 64 ± 17 % of recombinant Factor IX bound to the conformation-specific antibodies, and 9.4 ± 0.7 Gla residues were found (compared with 12 Gla in plasma FIX). When the 18-residue propeptide was deleted, secreted FIX contained no detectable native FIX antigen and no detectable Gla. Similarly, point mutations leading to substitution of Ala for Phe at residue -16 or Glu for Ala at residue -10 led to secretion of FIX containing 2% and 6% native antigen, respectively, and approximately 1-2 Gla residues. The molecular weight of each of the reoombinant FIX species, as estimated by SDS-PAGE, was identical to that of plasma FIX. NH2-terminal sequence analysis of the mutant FIX speoies yielded the NH2-terminal sequence of plasma FIX. These data indicate that the mutations made in the propeptide did not interfere with intracellular proteolytic prooessing of FIX. We conolude that the FIX propeptide participates in defining a recognition site that designates an adjacent glutamic acid-rich domain for γ-carboxylation. The association of the propeptide with the γ-carboxylation recognition site provides the first demonstration of a specific function served by a propeptide in post-translational protein processing.
EXPRESSION IN MAMMALIAN CELLS OF FUSION PROTEINS BETWEEN HUMAN FACTORS IX AND VII
Authors: Berkner K L , Busby S J , Gambee J , Kumar A
Published: August 2018

The vitamin K-dependent plasma proteins demonstrate remarkable similarities in their structures: all have multiple domains in common and extensive homology is observed within many of these domains. In order to investigate the structure-function relationship of these proteins, we have interchanged domains of one protein (factor IX) with that of another (factor VII) and have compared the expression of these fusion proteins with recombinant and native factors IX and VII. Oligonucleotide-directed mutagenesis was used to generate four fusion proteins: factor IX/VII-1, which contains the factor IX leader and gla domain fused to the growth factor and serine protease of factor VII; factor VII/IX-1, a reciprocal fusion protein of factor IX/VII-1; factor IX/VII-2, which contains the factor IX leader adjoined to the mature factor VII protein sequence; and factor VII/IX-2, the reciprocal fusion protein of factor IX/VII-2. The cDNAs encoding all four proteins were cloned into mammalian expression vectors, and to date three of these (factors IX/VII-1, 2 and VII/IX-1) have been transfected into baby hamster kidney (BHK) cells or 293 cells and characterized. Factors IX/VII-1 and VII/IX-1 were both secreted at levels comparable to recombinant factors IX and VII. The factor IX/VII-1 was identical in molecular weight to native or recombinant factor VII (i.e., 53 K). Factor VII/IX-1 was expressed as two proteins with molecular weights around 68 kd, as observed with recombinant factor IX. The factor IX/VII-1 protein has been purified to homogeneity and has been found to possess factor VII biological activity, but at a specific activity approximately 20% that of plasma factor VII. Thus, the gla domain of one clotting factor is capable of directing the activation of another and of generating biologically active protein. In contrast, no activity was observed with the factor IX/VII-2 fusion protein, indicating that there are limits to the interchanges which can generate functional blood clotting factors.
DOSE-FINDING STUDY OF THROMBOLYSIS IN ACUTE MYOCARDIAL INFARCTION BY SINGLE-CHAIN PROUROKINASE
Authors: Diefenbach C , Erbel R , Meyer J , Hamm C W , Schofer J , Mathey D G
Published: August 2018

In 24 patients (pts)(mean age 54.2 ± 12.1 years) with acute myocardial infarction (onset of symptoms <6 hrs; mean duration 181.8±54.4 minutes) single-chain prourokinase (PUK) was applied intravenously. PUK is an inactive proenzyme of human urokinase isolated from recombinant Escherichia coli. Efficiency and safety of low and high dose application were analysed. Complete coronary artery occlusion was proved before start of thrombolysis therapy. After 5000 IU heparine intravenously the first 12 pts were given 10 mg as a bolus followed by 30 mg within 1 hour (group A), the next 12 pts received the double dose: 20 mg as a bolus and 60 mg within 1 hour (group B). After PUK infusion 20 IU/ kg heparine were administered continously in all pts for 72 hours. There were no significant differences in age, bodyweight and duration of symptoms in both groups In group A in 6 of 12 pts (50%) reperfusion of the occluded coronary artery was achieved within 58.3±17.2 minutes; in group B 9 of 12 (75%) vessels reopened within 44.0±29.9 minutes. The remaining stenosis of coronary artery was 91% in both groups. In group A no drop of fibrinogen or plasminogen was observed; only 2 pts of group B showed significant drops of fibrinogen (below 100 mg%) and plasminogen (below 40%) plasma level. In no patient major bleeding or any other acute adverse effect occurred.

Thus, intravenous application of 80 mg PUK is more effective than 40 mg PUK with low incidence of systemic side effects.
CEREBRAL THROMBOLYSIS WITH INTRAVENOUSLY ADMINISTERED RECOMBINANT LOW- MOLECULAR-WEIGHT-UROKINASE AND RECOMBINANT PRO-UROKINASE IN A DOG MODEL
Authors: Hirschberg M , Manoutchei A , Klemens B , Hofferberth B
Published: August 2018

There is increasing evidence that recombinant prourokinase (rec-pro-UK) is a proenzyme which in vivo systems may induce activation of the fibrinolytic system with a better thrombus selectivity than that obtained with active urokinase.

In order to study the effects of rec-pro-UK and low-molecular-weight-urokinase (LMW-UK) on acute stroke, a thrombus was induced in the middle cerebral artery (MCA) of anesthetized mongrel dogs (n=12). Occlusion of the vessel was confirmed by angiography. Following a 1 hour period of MCA occlusion, in six animals LMW-UK was administered intravenously at a dose of 4000 lU/kg/min. Angiographically confirmed thrombolysis occurred after 30 minutes. Thrombolysis by LMW-UK was accompanied by bleeding from all surgical wounds and consumption of plasminogen, alpha-2-antiplasmin and fibrinogen. Rec-pro-UK was administered to six other dogs in a LMW-UK-equivalent dosis. Thrombolysis was achieved after 30 minutes in all six cases without inducing a systemic lytic state. Neither in the LMW-UK-group nor in the group treated with rec-pro-UK intracerebral bleeding complications were observed on post mortem examination.

Our findings indicate that intravenous administration of rec-pro-UK - because of the lack of systemic side-effects - may be a safe way of rapid thrombolysis of occluded cerebral arteries in acute stroke.
INTRAVENOUS THROMBOLYTIC THERAPY WITH SINGLE-CHAIN UROKINASE-TYPE PLASMINOGEN ACTIVATOR IN PATIENTS WITH ACUTE MYOCARDIAL INFARCTION
Authors: Bode C , Schwarz F , Schuler G , Zimmermann R , Kubler W
Published: August 2018

Seventeen patients with acute myocardial infarction were treated with heparin combined with intravenous single-chain urokinase-type plasminogen activator (scu-PA),obtained from transformed human kidney cells. 4 patients in group I and 13 patients in group II received a bolus of 4 mg and 7-5 mg followed by an infusion of 11 mg and 40.5 mg over 60 minutes, respectively. Thrombolysis was achieved in no patient of group I during intravenous infusion of scu-PA, however, upon subsequent intracoronary infusion of 250000 IE streptokinase, reperfusion could be established in two out of three patients. In group II seven patients were successfully treated with intravenous infusion of scu-PA. In 2 of the 6 patients unsuccessfully treated with intravenous scu-PA,intracoronary streptokinase was subsequently administered. In both cases recanalization could not be achieved. The effects on the hemostatic system are summarized below. In all patients a severe residual stenosis persisted after thrombolytic treatment and 13 patients underwent PTCA.

Group Reperfusion Fibrinogen Plasminogen Antiplasmin

There was no difference between sucessfully and unsuccessfully treated patients in group II with respect to the effect of treatment on serum parameters.

It is concluded that intravenous infusion of scu-PA at a dose of 15 mg over 60 minutes is ineffective treatment for patients with acute myocardial infarction. However,at a dose of 48 mg over 60 minutes this form therapy is effective, save and specific.
DIPYRIDAMOLE ALONE OR WITH LOW DOSE ASPIRIN DOES NOT PREVENT ACUTE PLATELET THROMBUS FORMATION IN STENOSED DOG CORONARY ARTERIES
Authors: Folts J D , Smith S R
Published: August 2018

Dipyridamole (Dip) is reputed to inhibit (I) platelet aggregation (PA) and acute thrombus formation (ATF) by two mechanisms including inhibiting 1.) platelet (Pt) phosphodiesterase, 2.) adenosine (A) reuptake by red cells, which should raise plasma A. Both effects should raise Pt cyclic AMP and thus be a potent platelet inhibitor (PI). Because aspirin (AS) inhibits Pt thromboxane A2 production, a synergistic (S) PI effect for ASA and Dip given together has been postulated and used in clinical trials but this S has never been shown to I ATP in any in vivo model, which reasonably mimics human arterial stenosis. We have shown that ATF followed by embolization, occurs periodically in mechanically stenosed (MS) monkey and rabbit carotid arteries, and dog (D) and pig coronary arteries (CA), causing cyclical reductions in coronary blood flow (CRF) (measured with EMF probes) and periodic acute ischemia, and that these CRF can be abolished with a variety of PI including 3.0 mg/kg of ASA. To determine if there is a S effect between ASA and Dip, in open chest D, Dip was given, 2.0 mg/kg IV to D with a MS circumflex CA and having 14±5 CRF’s per hour, due to periodic ATF; and simultaneously flow measured in an unstenosed normal LAD CA. The frequency and size of CRF’s were not changed by Dip, although ABP decreased 21±9 mm Hg and blood flow in the unstenosed LAD increased 259±47%. A low dose of ASA, 1.0 mg/kg, which by itself diminishes but does not abolish CRF’s in this model was given IV 10 min. after Dip and CRF’s continued unchanged. When a second dose of ASA 1.0 mg/kg was given IV to reach the minimum effective dose of ASA in this model, CRF were abolished in all D. Thus Dip was not effective alone or in combination with low dose ASA to I CRF in this model which simulates the patient with stenosed CA. The majority of clinical trials that show inhibition of ATF, used ASA and Dip together without 3 separate patient groups on Dip alone, ASA alone and ASA plus Dip. The widespread use of Dip with ASA to prevent ATF in man needs to be reevaluated.
THE BEHAVIOUR OF RABBIT PLASMINOGEN AT THE LUMINAL SURFACE OF RABBIT AORTA IN VIVO BEFORE AND AFTER BALLOON-CATHETER INJURY
Authors: Hatton Mark W C , Moar Susan , Richardson Mary
Published: August 2018

A previous study from this laboratory has identified the susceptibility of the de-endothelialised aorta, particularly the proteoglycan (PG) components of the extracellular matrix (ECM), to proteolytic damage if exposed to plasmin in vitro. To explore the possiblity that this occurs in vivo, a possible association between 125I-plasminogen (PLG) binding to the arterial wall, its activation to plasmin (PLN) and, subsequently, proteolytic damage to the intimal ECM has been studied. Intravenous injection of 125I-PLG in healthy N.Z. white male rabbits showed that PLG associated minimally (<0.01% of circulating PLG/cm2 /ml blood at 1 h) with the thoracic aorta endothelium, measured after Hautchen preparation from 1-cm vessel segments. Trans endothelial passage, measured as 125I-PLG associated with thg subendothelium (intima-media), progressed to 0.015%/cm2 /ml blood at 1 h. In contrast, the process of de-endothelialisation by balloon catheter led to a rapid uptake of bI-PLG by the denuded vessel surface. At saturation (approx. 10 min after injury), 0.7 - 0.8% of circulating PLG/cm2/ml blood was adsorbed by the entire de-endothelialised intima-media: Of the adsorbed PLG, 2-3% was associated with the platelet layer. Uptake was not inhibited by eACA (dose: 200 mg/kg) given i.v. before I-PLG. Adsorbed PLG was not released significantly from segments incubated in MEM containing 4% (w/v) RSA in vitro PLN activity was not detected. Furthermore, assessment of the ECM by transmission electron microscopy, after ruthenium red staining, showed that uptake of PLG by the de-endothelialised vessel in vivo was not associated with obvious damage to the PG components. Supported by the Heart and Stroke Foundation of Ontario.
MAY LMW (CY 216) BE ADMINISTERED DURING PREGNANCY ?
Authors: Forestier F , Daffos F , Rainaut M , Cornu P , Deschamps A , Toulemonde F
Published: August 2018

One of the main problems related to the use of fractionated heparin during pregnancy concerns its transplacental passage.

Previous studies showed LMW heparin fraction CY 216 has no teratogenic effects, and when labelled, does not cross the placental barrier in animal, and does not appear into the milk.

We studied the transplacental passage following subcutaneous administration of large dosage (17.500 AXa IC u) in 7 women who where going to have an abortion during the third trimester of gestation because of severe fetal malformation, after informed consent.

Blood samples were taken before and 3 h after injection from che mother , from the fetuses 3 h after mother injection -using ultrasound guidance of the needle and aspiration of blood in the umbilical vein.

Biological assays showed that the effects are clearly observable in mother, whereas no change was observed from the fetus.

Thus, it was justifiable to treat, for several reasons, 22 patients using CY 216 during a period of 2 to 5 weeks before delivery. Treatments were successful and no complication has been observed. The cord blood samples at birth never showed any biological activity.

These data seem to clearly indicate that there is no passage through the placental barrier of CY 216 which offers a new possibility of treatment during pregnancy.
CHARACTERIZATION OF THE GENE FOR HUMAN HISTIDINE-RICH GLYCOPROTEIN
Authors: Koide T
Published: August 2018

Human histidine-rich glycoprotein (HRG) is a single-chain glycoprotein in plasma which is considered to modulate a coagulation and fibrinolysis system with the ability to bind to heparin, plasminogen, fibrinogen, thrombospondin, etc. Recently we have elucidated the primary structure of HRG by determining the nucleotide sequence of its cDNA, and showed that HRG is composed of several different types of internal repeats, each one of which shows considerable homology with the functional and/or structural domains of other proteins including high molecular weight kininogen, antithrombin III, cystatins, and proline-rich protein and peptide. Thus, the multifunctional property of HRG was suggested to be due to its multi-domain structure. In the present studies, a human genomic DNA library, cloned in the bacteriophage vector Charon 4A, was screened for HRG gene using a full-length cDNA coding for human IMI as a probe. A total of 7 clones were isolated from 6 × 105 phage and each was plaque purified. The entire HRG gene is represented in 3 genomic inserts with overlapping sequences that carry human DNA spanning 30 kb. Overlapping gene fragments were subcloned into pUC9 and characterized by Southern blot hybridization using 5’ and 3’ end probes isolated from human HRG cDNA and by DNA sequencing. These studies have shown that the gene for human HRG spans about 9 kb and consists of at least 5 exons and 4 introns. The putative histidine-rich region consisted of 12 tandemly repeated sequences of a 5 amino acid segment and 2 proline-rich regions contiguous to it are likely to be involved within one exon.
IMMUNOASSAYS FOR SINGLE CHAIN URINARY-TYPE PLASMINOGEN ACTIVATOR (SCUPA) IN PLASMA AND IN CELL CULTURE SUPERNATANTS
Authors: Mahmoud M , Hammerschmidt F , Scheuerlein H , Gaffney P J
Published: August 2018

Monoclonal antibodies (mabs) to SCUPA have been generated in balb/C mice by conventional means and have been demonstrated to have no crossreactivity with two chain urinary-type plasminogen activator (TCUPA). These mabs have been used to develop two types of specific assay for SCUPA. Mabs coated on polyvinyl plates in conjunction with polyclonal antibodies (pabs) to TCUPA have allowed the development of a catcher-tag ELISA using alkaline phosphatase-label led goat anti-rabbit IgG as a final step. The sensitivity range of the assay was 0.5 - 10.0 iu/ml. A second bioimmunoassay (BIA) using SCUPA mabs as catcher and inplate development with glu-plasminogen and S-2251 has yielded an assay with a sensitivity range of 0.5 - 10.0 iu/ml. The international unitage ascribed in these assays was derived by comparing the hydrolysis of S-2444 by the I.S. for TCUPA with the purified SCUPA following full activation with plasmin.

Using these assays it was found that normal pooled plasmas contained about 1.0 iu of SCUPA antigen which was fully inhibited such that no activity was evident by the BIA assay for SCUPA. This suggests that urokinase in plasma is present in two forms: SCTJPA bound to inhibitor and TCUPA which is biologically active when assayed using a BIA based on immobilised pabs to TCUPA. Cell supernatants from cultured human lung fibroblasts yield a SCTJPA/TCUPA ratio of 70/30 using S-2444 chromogenic assay following a plasmin-mediated SCTJPA-TCUPA conversion step. It was also shown that, in these cell supernatants, SCTJPA was secreted with no inhibitor bound to it, since the BIA and ELISA data were quite similar. A curious feature of these assays, which is as yet unexplained, is the observation that urokinase (both plasmin activated SCTJPA and TCUPA), when immunologically adsorbed on to the PVC-immobilised specific mabs or pabs used in this study, readily activated plasminogen but showed no hydrolytic activity on the chromogenic substrate, S-2444.
ANTITHROMBOTIC PROPERTIES OF HEPARIN IN A NEONATAL MODEL OF THROMBIN INDUCED VENOUS STASIS THROMBOSIS
Authors: Schmidt B , Buchanan M R , Ofosu F , Brooker L A , Andrew M , Univ McMaster
Published: August 2018

Anecdotal clinical experience suggests that more heparin is required in newborn infants that in adult patients to effectively treat thrombotic disease. We compared the ability of heparin to inhibit thrombus formation induced by a pathological bolus of thrombin and stasis in newborn piglets and 3 week old pigs. The coagulation system of the newborn piglet closely resembles that of the human neonate Including low antithrombin III (AT-III) activity (0.5U/ml). By 3 weeks, adult porcine values for coagulation factors and inhibitors are reached, while blood volume/kg body weight still approximates that of the newborn piglet. Piglets and pigs were pretreated with saline, 10 or 25U/kg heparin (n ≥16/group/dose. Following an injection of 100U/kg thrombin, systemic 125I-fibrinogen consumption and local 125I-fibrinogen incorporation into jugular venous stasis thrombi were measured. Peak heparin levels were identical In both age groups (Anti-factor Xa assay and protamine sulphate titration). Heparin was less effective in preventing thrombus formation in piglets than in pigs (Table). Heparin was also less effective in preventing systemic neonatal 125I-fibrinogen consumption (p<0.0001 at both heparin doses).

Raising AT-III levels to adult values significantly improved the antithrombotic properties of heparin in neonatal piglets. Thrombus formation was completely abolished in 19 or 22 piglets who received a combination of human or porcine AT-III concentrate and 25U/kg heparin. Raising the heparin dose to 50U/kg had the same effect. We conclude that the efficacy of heparin in neutralizing thrombin is decreased in newborn piglets. Treatment with AT-III concentrate overcomes this relative heparin resistance, and may help reduce high heparin doses otherwise required in neonatal thrombotic disease.
FREQUENCY AND IMPLICATIONS OF SEVERE NEONATAL PROTEIN C DEFICIENCY
Authors: Manco-Johnson M J , Abshire T C , Jacobson L J
Published: August 2018

The newborn infant has a physiologically low level of protein C which rises very slowly in postnatal life. The frequency and significance of severe neonatal protein C deficiency has not been reported. In this study, protein C levels were measured in 110 newborn infants at the time of birth using functional (amidolytic, Cact) and immunologic (Laurell rocket, Cag) assays. The protein C levels were compared with a marker of thrombin activation (D-dimer fragment of fibrin, +D-D) and infants were subsequently followed for signs and symptoms of thrombosis. Results are summarized below (protein C levels are expressed as U/ml).

Thirteen infants had protein C levels compatible with the homozygous deficiency state. Extremely low levels of protein C (<0.20 U/ml) were not found in well term infants and were rarely noted in stable preterm infants. D-D were infrequently present and no thrombosis occurred. Near term infants born with fetal distress frequently showed +D-D but rarely demonstrated extremely low levels of protein C. None of these infants required indwelling arterial catheters and no thromboses occurred. Preterm infants with severe respiratory distress showed lower protein C levels at birth (p <0.01). Although 71% had +D-D, thromboses in these infants were all related to invasive catheterizations. In contrast, the study population of twins demonstrated a high frequency of severe protein C deficiency with negative D-D and frequent thromboses, three of which occurred in the absence of instrumentation. In summary, severe protein C deficiency and thrombin activation are common in sick preterm infants with the risk of thrombosis increased by intravascular catheterization. In contrast, twins with severe protein C deficiency may manifest a thrombotic risk which is independent of thrombin activation or catheterization.
EFFECT OF ANTIPLATELET THERAPY ON EARLY GRAFT PATENCY AFTER CORONARY ARTERY BYPASS GRAFTING: VA COOPERATIVE STUDY # 207
Authors: Goldman S , Copeland J , Moritz T , Henderson W , Harker L A
Published: August 2018

To determine if specific antiplatelet therapy improved graft patency after coronary artery bypass grafting (CABG) -we compared (1) aspirin(325 mg qd), (2)aspirin(325 mg tid),(3) aspirin and dipyridamole(325 mg and 75 mg resp.tid), (4) sulfinpyrazone(267 mg tid) and (5) placebo(tid).Therapy was started 48 hours before CABGexcept for aspirin. When aspirin was a treatment,one 325 mg dose was given12 hours before surgery. Graft patency data were obtained early, one week, and then later, one year, after surgery. Preliminarydata, based on local interpretation of the angiograms at each center, in the firs 496 patients (1711 grafts), revealed thefollowing early graft patencies: aspiri qd (93%), aspirin tid (93%), aspirin and dipyridamole (93%),and sulfinpyrazone (92%). All these therapies improved(P<0.005)early graft patency compared to placebo (84%). Chest tube drainage measured within the first 35 hours after CABG revealed that the median loss with aspirintid (1114 ml) and aspirin and dipyridamole (972 ml) exceeded (P<0.001) placebo (802 ml) while aspirinqd (880 ml) and sulfinpyrazone (750 ml) did not. The reoperation rate was greater(P<0.01) in all the treatment groups thatcontained aspirin (6.1%) compared to the two non aspirin groups (1.9%). Overall operative mortality was 2.1%. In conclusion, graft patency was improved early after CABG with antiplatelet therapy. Two regimens which included preoperative aspirin has increased blood loss after CABG and preoperative aspirin increased the reoperation rate.
THE PHOSPHOLIPID-BINDING SITE OF FACTOR VIII IS LOCATED ON THE 80 kD LIGHT CHAIN
Authors: Kemball-Cook G , Edwards S J A , Sewerin K , Andersson L-O , Barrowcliffe T W
Published: August 2018

The binding of Factoi. VIII (F.VIII) peptides to phospholipid (PL) vesicles has been studied by two different methods involving the use of fractionated anti-F.VIII:C I-Fab123’pre
viously reported, i-Fab123’ was fractionated by immunoadsorptionwith F.VIII-PL complexes into two pools:one binding only to PL-binding sites on F.VIIIsAg (PL-site antibody), the other directed against other antigenic sites (non-PL-site antibody).

The first technique used was a modification of the method of Weinstein et al. (Proc.Natl.Acad.Sci.USA, 78, 5137-5141, 1981), and involved incubation of the two anti-F.VIII pool swith F.VIII-containing samples, followed by electrophoretic separation of the complexes on the basis of size in non-denaturing SDS gels: this technique allows qualitative analysis of antibody reactive peptides in highly impure samples. Non-PL-site pool reacted with a range of peptides with MrMapparent Mr 90 kD up to 280 kD, a similar pattern to that of ’heavy chain’(HC) peptides of F.VIII seen on SDS-PAGE under reducing conditions; the PL-site antibody, however, reacted only with peptides at apparent Mrs of 80 kD and sometimes150 kD, but not with bands of higher Mr a pattern more consistent with binding to light chain (LC) peptides. Thesame patterns with the two labels were seen in both plasma and F.VIII concentrate

The second approach employed the two labels described above in direct immunoradiometric assays (IFMA’s) on purified human F.VIII peptides prepared by immunoaffinity chromatography and ion exchange on Mono Q gel. Both PL-site and non-PL-site labels measured similar amounts of F.VIII m a sample containing both HC and LC peptides; however, on assaying a sample containing purified HC peptides alone, PL-site antibody measured only 2% of F.VIII:Ag found by non-PL-site label, indicating that PL-binding sites present in samples containing both HC and LC are absent in HC alone.

Results from both these immunological methods indicate that the 80 kD LC peptide of F.VIII carries the PL-binding site.
THE INTERACTION OF PURIFIED FACTOR VIII WITH PLATELETS
Authors: GanZ P R , Tackberry E S , Rock G
Published: August 2018

Factor VIII is known to interact with Factors IXa and X to generate activated Factor X. A requirement for phospholipid in this reaction suggests that this "tenase" protein complex is assembled on a membrane surface. As a first step in studying the involvement of Factor VIII in this process, we wished to determine whether purified Factor VIII could interact directly with platelets. Factor VIII utilized in these experiments was purified from heparinized blood by a six-stage procedure including cryoprecipitation, polyethylene glycol precipitation, Affi-Gel Blue, Aminohexyl, polyelectrolyte E5 and immunoaffinity chromatography. This yielded a single-chain high molecular weight species of approximately 260,000 (specific activity 5,200 units/mg). This homogeneous protein was then radiolabelled with Na125I by a procedure which allowed the retention of approximately 60-80% of the procoagulant activity of Factor VIII. The kinetics of binding of 125I-Factor VIII to washed platelets at physiological concentration (approximately 3xl08/mL) was examined. Our results showed that for Factor VIII concentrations between 0.38 and 3.0 ng/mL there was a linear uptake of radiolabelled Factor VIII, whereas for concentrations above 10ng/mL only a slight increase in uptake occurred. To further define the association of purified Factor VIII with the platelet membrane, we also labelled Factor VIII with a bifunctional, photoactivatable cross-linking reagent, N-[4-(p-azido-m-[125]iodophenylazo)benzoyl]3-aminopropyl-N1 -oxysuccinimide ester. Analysis by PAGE showed thatthis reagent reacts predominantly with residues in the light chain or neahe C-terminal portion of Factor VIII. When mixed with thrombin-stimulated platelets, the cross-linked Factor VIII molecule was shown to transfer greater than 80% of the 125I label to a polypeptide of M.W. 80,000-90,000 isolated from platelet lysates. Autoradiographs of the labelled platelet preparations demonstrated that other minor polypeptides were radiolabelled. These experiments suggest that Factor VIII interacts closely with a platelet membrane protein which could represent a binding site for Factor VIII
APSAC versus placebo: A Multicenter Study of Safty and Early Morality in Acute Myocardial Infarction
Authors: Julian D G , Borthwick L S , Reid D , Jennings K P , Wainwright R J , Rodger J C , Wood D , Flax J S , Phillips W S
Published: August 2018

A prelimiry study was carried out of obtain data on mortality,benefit and the safety of anisoylated plasminogen streptokinase activator complec (APSAC).90 patients from 7 centers,with symptons of acute myocardial infarction of not more than 4 hours duration,were randomised to receiveeithere 30U of APSAC as an intravenous injection over 4 to 5 minutes or placebo given by the same route.45 patients recevied APSAC and placedo,mean time to treatment was 3 hours 20 minutes and 3 hours, respectively.

Mortality at 30 days was 7 deaths in theplacebo group and 1 death in the APSAC group (p=0.058),these were all related either directly or indirectly to the infarct:cardiogenic shock (2),ventricular asystole(2) further acute myocardial infraction(2),ventricular fibrillation (1) and pulmonry embolism (1).The patient who died after APSAC therany had presented with an anterior infraction; 5 patients who died inthe placebo group presented with anterior infarcts and 2 with inferior infarcts. Most daeths occured in the group randomised 2 hours 30 minutes to 4 hours post-infarcation.

Both systolic and diastolic blood pressure were generally similar in each groupfrom 1 hour to 1 month after treatment. 35 percent of petients in the placebo group experienced cardiovasular events (2 of whom died from cardiogenic shock) compared with 20 percent of APSAC of treated patients (1 of whom died from pulmonary embolism).Minor haematuria was apparent in a grater number of patients in the APSAC group after 12 hours, but there was no differnce between groups after 1 or 2 weeks.

The result of this study ,although encouraging,should be interpreted with caution,mainly due to the small numbers of patients enrolled.There was however a trend towards incresed survival in the APSAC treated group (97.8 per cent) compared with84.4 cent survival in the placenbo groupat 30 days post-infarction.
EXPRESSION OF A PLATELET GLYCOPROTEIN Ibα RELATED PROTEIN IN THE HUMAN HISTIOCYTIC CELL LINE U937
Authors: Wicki A N , Kieffer N , Clemetson K J
Published: August 2018

It has previously been shown that platelet glycoprotein lb is expressed in a minority of cells of the human leukemic cell line HEL (Tabilio et al., EMBO J. 3, 453-459). The present study was performed to determine wheter U937 cells also share membrane proteins with platelets. Indirect immunofluorescence on histiocytic U937 cells with F(ab)2 fragments of polyclonal anti GPIb antibodies and with different monoclonal antibodies revealed a uniform staining of the cell surface. A single polypeptide chain of apparent molecular mass 60,000 was precipitated under reducing and nonreducing conditions from nonanoyl-N-methyl glucamide solubilized, surface labelled and metabolically labelled U937 cells by the different antibodies. Competitive immunoprecipitation performed in the presence of an excess amount of unlabelled platelet glycocalicin, a large fragment of GPIbα, completely displaced the 60 kDa polypeptide. We were unable to demonstrate the presence of a polypeptide corresponding to the α-subunit of GPIb. Therefore, we conclude that U937 cells produce a 60,000 Da protein which is immunologically closely related to platelet GPIbα
NEUTRALIZATION OF HEPARIN-LIKE SACCHARIDES BY COMPLEMENT S PROTEIN/VITRONECTIN
Authors: Lane D A , Flynn A M , Pejler G , Lindahl U , Preissner K
Published: August 2018

S protein, a major inhibitor of the assembly of the membrane attack complex of complement, has recently been shown to be identical to the serum spreading factor vitronectin. It has also been demonstrated to have anti-heparin properties. We have studied the heparin neutralizing properties of S protein/vitronectin using heparin, heparan sulfate and heparin oligosaccharides with high affinity for antithrombin. The ability of heparin fractions, Mr 7300-18800, and of the International Heparin Standard, to accelerate the inactivation of thrombin and Factor Xa by anti thrombin was readily neutralized by S protein/vitronectin. Addition of the protein to the various saccharide fractions at a molar ratio 1-3/1 produced 50/ neutralization, while complete neutralization was achieved at a molar ratio of <10/1. Moreover, S protein/vitronectin efficiently neutralized oligosaccharides of Mr 2400-7200, unlike the two other physiologically occur ing heparin neutralizing proteins histidine-rich glycoprotein <HRG) and platelet factor 4 < PF4) <Lane et al (1986) J. Biol. Chem.261, 3980; Lane et al (1984) Biochem. J. 218, 725). Like PF4, but unlike HRG, S protein/vitronectin readily neutralized the anticoagulant activities of heparan sulfate of Mr ˜20000. These findings indicate that S protein/vitronectin requires little more than the antithrombin-binding pentasaccharide with which to interact in order to express its anti-heparin activity. Furthermore, the results suggest that S protein/vitronectin may be a physiological1y important modulator of the anticoagulant activity of heparin-like material on or near the vascular endothelium.
PROTEIN C, PROTEIN S AND THE FIBRINOLYTIC SYSTEM IN PATIENTS WITH A HISTORY OF THROMBOSIS
Authors: Malm J , Laurell M , Nilsson I M , Dahlbäck B
Published: August 2018

Consecutive patients with a history of thrombo-embolic disease (n = 241, 109 males, 132 females, mean age 46 y), referred to the Coagulation Laboratory during an 18 month period, were analysed for defects in their coagulation and fibrinolytic systems. The diagnosis of thrombosis had been verified with phlebography and that of pulmonary embolus with scintigraphy or angiography. Retinal venous thrombosis was found in 15 of the patients. In 15 cases the thrombotic episodes occurred postoperatively, in 15 during pregnancy, in 12 during the postpartum period and in 20 during use of oral contraceptives. In the remaining cases no clinical riskfactors were identified.

The concentration of protein C zymogen was measured with an immunoradiometric assay. Functional protein C was determined with a clotting inhibition assay. Protein C deficiency was found in 8 cases. Two of these had a functional protein C deficiency with normal zymogen levels. The concentration of total, as well as free (not in complex with C4b-binding protein), protein S was determined with a radioimmunoassay. Two cases of protein S deficiency were detected. Three patients with antithrombin III deficiency and two with plasminogen deficiency were found.

The fibrinolytic activity after venous occlusion was analysed in 216 patients. Decreased levels were found in 32 %. The concentration of tissue plasminogen activator inhibitor (PAI) was measured in 110 patients and found to be increased in 65 % of the cases. In 99 patients both the fibrinolytic activity and the PAI concentration were measured. A combination of decreased fibrinolytic activity and increased levels of PAI was found in 44 cases. The concentration of tissue plasminogen activator antigen was decreased in 22 % of 105 cases analysed.

Thus, in this material of patients with thrombo-embolic disease, abnormalities were found in 47 %. Defects in the fibrinolytic system were the most common findings. Protein C or protein S deficiency was diagnosed in less than 5 % of the cases.
MONOCLONAL ANTIBODIES THAT RECOGNIZE Ca2+-INDUCED CONFORMER OF PROTEIN C, INDEPENDENT OF GLA RESIDUES
Authors: Sugo T , Tanabe S , Shinoda K , Matsuda M
Published: August 2018

Monoclonal antibodies (MCA’s) were prepared against human protein C (PC) according to Köhler & Milstein, and those that recognize the Ca2+-dependent PC conformers were screened by direct ELISA in the presence of 2 mM either CaCl2 or EDTA. Out of nine MCAߣs thus screened, five MCA's designated as HPC-1˜5, respectively, were found to react with PC in the presence of Ca2+ but not EDTA. By SDS-PAGE coupled with Western Blotting performed in the presence of 2 mM CaCl2, we found that two MCA’s HPC-1 and 2, recognized the light chain, and two others, HPC-3 and 4, recognized the heavy chain of PC. But another MCA, HPC-5 was found to react with only non-reduced antigens. Further study showed that HPC-1 and 5 failed to react with the Gla-domainless PC, i.e. PC from which the N-terminal Gla-domain of the light chain had been cleaved off by α-chymotrypsin. However, all the other three MCA's retained the reactivity with the antigen in the presence of Ca2+ even after the Gla-domain had been removed. The binding of these MCA’s to PC in the presence of Ca2+ was found to be saturable with respect to the Ca2+ concentration and the half maximal binding for each MCA was calculated to be about 0.5+mM. Moreover, many other divalent cations such as Mg2+, Mn2+ , Ba2+, Zn2+, Co2+, Sr2+, were found to substitute for Ca2+ in inducing the metal ion-dependent but Gla-domain-independent conformer of PC.

Cross-reactivity to other vitamin K-aependent plasma proteins was examined by direct ELISA; HPC-2 and 3 reacted solely to PC, but HPC-1 and 4 also reacted with prothrombin and HPC-5 with both prothrombin and factor X.

These findings indicated that there are two or more metal binding sites besides the Gla-domain, possibly one in the light chain and the other(s) in the heavy chain. The presence of these metal binding sites may contribute to the unique conformer of vitamin K-dependent plasma proteins including protein C.
CLINICAL APPLICATIONS OF THE MEASUREMENT OF CROSSLINKED FIBRIN DEGRADATION PRODUCTS FOLLOWING MYOCARDIAL INFARCTION
Authors: Rowbotham B , Whitaker A N , Bett J D N , Woodhouse S P , Bunce I H , Elms M J , Masci P , Webber A J
Published: August 2018

Crosslinked fibrin degradation products (XLFDP) were measured serially in 135 consecutive admissions to a coronary care unit. Plasma was assayed by ELISA using the monoclonal antibody DD-3B6/ 22 which recognizes a site near the γγ crosslink. There was no significant difference between initial levels in patients with acute myocardial infarction, angina or noncardiac chest pain. Increased initial levels were associated with severe congestive cardiac failure (p< 0.001) and thromboembolic events (p<0.05) but did not correlate with survival or infarct size as evaluated by peak creatine kinase level. 44% of patients developed significantly increased levels (>400 ng/ml) without explanation at some time in the 10 days following infarction. Increased levels of XLFDP are a sensitive marker of intravascular thrombosis and we postulate that occult thromboembolic disease occurred in some of these patients. In a subsequent cohort of 42 patients wtih Q-wave infarcts admitted to the ISIS 2 Trial, treatment with intravenous streptokinase (SK:1.5 x 106U) in 21 patients caused plasma levels of XLFDP to rise to variable levels (range 400 - 10,000 ng/ml) compared to no rise in those in the placebo group. Positive Dimertest titres also occurred after SK in 15 cases, but remained uniformly negative in the controls. It is not clear whether coronary thrombolysis alone can explain these elevations accompanying SK administration. Other potential sources of XLFDP which should be considered are mural thrombi, pulmonary emboli, venous thrombi and circulating XL fibrin complexes. The importance of the last is being investigated. Measurement of XLFDP requires further evaluation as a monitor of coronary thrombolysis but may play a useful role in screening patients at risk of cardiac failure or thromboembolism
LUPUS ANTICOAGULANT AND REPEATED ABORTIONS: A CASE- CONTROL STUDY
Authors: Barbui T , Cortelazzo S , Galli M , Parazzini F , Radici E , Rossi E
Published: August 2018

In the last few years a role of Lupus Anticoagulant (LAC) in the aetiology of repeated spontaneous abortions and intrauterine deaths has been repeatedly suggested. To quantify this association few da ta are available, since the published reports are generally based on uncontrolled and small clinical series. We have analyzed data from a case-control study conducted in Bergamo and Milan, two contiguous provinces in Lombardia, Italy. Cases were 63 women, mean age 30 years, range 23-40, with 2 or more "sine causa" spontaneous abortions (repeated abortions) admitted between March 1985 and December 1986 to the Ospedali Riuniti of Bergamo and Istituti Clinici di Perfezionamento of Milan. Controls were 63 women, mean age 32 years, range 20-49, with 1 or more live births and without spon taneous abortions, admitted to the same Institutions for neither gynaecological nor cardiovascular acute conditions. Informations were collected on sociodemographic factors, gynaecological and obstetrical data and related medical history. LAC was diagnosed according to the Working Party reccomandations (1983) and Systemic Lupus Erythematosus (SLE) according to the revised criteria of ihe American Rheumatism Association (1982). 11 out of 63 cases (17%) (95% confidence interval ranging from 9.5% to 34% based on the Poisson's approximation) were LAC positive, whereas in none of 63 controls this inhibitor was detected (X2
1adjusted for age = 10.1, p= 0.02). Similarly SLE was diagnosed in 4 cases (all having a Lupus Anticoagulant) and in none control (x2
1adjusted for age= 4.17, p=0.02). These findings confirm that LAC is associated with a positive history of repeated abortions, being present in about 10% of the cases. Conclusive estimate of relative risk is prevented by the small control gr'oup size (i.e. lack of positivity for LAC in controls), but very elevated risk (many tenfold increase) is sugge. sted.