bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure of the Human Factor VII Gene
Authors: O'hara Patrick J , Grant Frank A , Betty A , Haldmen J , Murray Mark J
Published: August 2018

Factor VII is a member of a family of vitamin K-dependent, gamma-carboxylated plasma protein which includes factor IX, factor X, protein C, protein S and prothrombin. Activated factor VII (factor Vila) is a plasma serine protease which participates in a cascade of reactions leading to the coagulation of blood. Two overlapping genomic clones containing sequences encoding human factor VII were isolated and characterized. The complete sequence of the gene was determined and found to span 12.8 kilobases. The mRNA for factor VII as demonstrated by cDNA cloning is polyadenylated at multiple sites but contains only one AAUAAA poly-A signal sequence. The mRNA can undergo alternative splicing forming one transcript containing eight segments as exons and another with an additional exon which encodes a larger pre-pro leader sequence. The portion of the pre-pro leader coded for by the additional exon has no known counterpart in the other vitamin K-dependent proteins. The positions of the introns with respect to the amino acid sequence encoded by the eight essential exons of factor VII are the same as those present in factor IX, factor X, protein C and the first three exons of prothrombin. These exons code for domains generally conserved among members of this gene family, including a pre-pro leader (the essential exon la and alternative exon lb), a gamma-carboxylated domain (exons 2 and 3) a growth factor domain (exons 4 and 5) an activation region (exon 6) and a serine protease (exon 8). The corresponding introns in these genes are dissimilar with respect to size and sequence, with the exception of the third intron in factor VII and protein C. Four introns and a portion of exon 8 in factor VII contain regions made up of tandem repeats of oligonucleotide monomer elements. More than a quarter of the intron sequences and more than a third of the 3' untranslated portion of the mRNA transcript consist of these minisatellite tandem repeats. This type of structure is responsible for polymorphisms due to allelic variation in repeat copy number in other areas of the human genome. Tandem repeats can evolve as a result of random crossover in DNA whose sequence is not maintained by selection. This suggests that much of the sequence information present in the introns and untranslated portion of the message is dispensable.
EFFECT OF DIFFERENT HEPARINS ON THROMBOLYSIS WITH t-PA AND scu-PA IN RABBITS WITH EXPERIMENTAL THROMBOSIS
Authors: Juhan-Vague I , Stassen J M , Alessi M C , Kieckens L , Collen D
Published: August 2018

Infusion of heparin or low-Mr heparin fractions in animals with experimental thrombosis or in patients with thromboembolic disease may result in a significant reduction of the thrombus size, without being associated with measurable changes in the blood fibrinolytic parameters.

We measured the effect of clinical grade heparin (Hep) and of two low-Mr heparin fragments (CY216 and CY222 from Choay, Paris, France) on thrombolysis with t-PA and with scu-PA in a rabbit jugular vein thrombosis model (Collen et al., J. Clin. Invest. 71, 368, 1983). After thrombus formation, t-PA (0.25 mg/kg) or scu-PA (0.5 mg/kg) were infused over 4 hours. The heparins were administered at hourly intervals at the start and during the infusion as bolus injections of the following amounts (expressed in anti-Xa units): Hep: 70 (A) or 200 (B); CY216: 30 (A) or 90 (B); CY222: 50 (A) or 150 (B). Results were (mean ± SEM):

It is concluded that at sufficiently high doses, heparin and to a larger extent the two low-Mr heparin fractions CY216 and CY222, potentiate thrombolysis by rt-PA and scu-PA in this animal model of thrombosis.
PGI2 AND PGE1 INHIBIT SMOOTH MUSCLE CELL PROLIFERATION MITOTIC ACTIVITY AND EXTRACELLULAR MATRIX FORMATION
Authors: Sinzinger H , Zidek T , Fitscha P , Wagner O , Rogatti Waltraud
Published: August 2018

Earlier findings that activated smooth muscle cells (SMC) generate more PGI2 indicate a pathophysiological role of PG's in SMC-proliferation. In a rabbit experiment enhanced mitotic activity (MA) induced by ACTH or desoxycorticosterone acetate(DCS) is significantly depressed by both PGI2 and El.

most effective.Similarly, a decrease in collagen and glycosamino-glycan-formation canbe noted. PGI2 is able to inhibit quite selectively liberation of platelet derived growthfactor (PDGF). PDGF itself stimulates vascular PGI2-generation,inhibiting further PGDF-relese long-1lasting intermittent alternating PGI2/PGE1 therfore be of optional effect concerning the local vascular SMC-action
DECREASE OF PLATELET AGGREGATION AFTER INTAKE OF SMALL AMOUNTS OF PURIFIED EICOSAPENTAENOIC ACID IN ELDERLY PEOPLE. POSSIBLE ROLE OF VITAMIN E
Authors: Croset M , Vericel E , Rigaud M , Coupron Ph , Dechavanne M , Lagarde M
Published: August 2018

Platelet hyperactivity is believed to be involvedin the thrombotic complications of elderly people.Since it has been proposed that eicosapentaenoic acid(EPA) can reduce platelet functions in normal humans,we have investigated its effect in elderly people. The daily intake (100 mg EPA) was at least 20 fold lower than usually tested. In a randomized, double blind study, eight people ingested EPA given as a pure triglyceride (1,3-didecanoyl,2-eicosapentaenoyl-glycerol) for 2 months and eight people ingested a placebo containing the same amount of vitamin E than in EPA preparation. Platelet aggregation, the oxygenatedmetabolism of arachidonic acid (AA), and vitamin E content of both plasma and platelets were investigated. Ingestion of EPA resulted in a slight but significant reduction of platelet-rich plasma aggregation in response to epinephrine and AA. The same decrease was observed with washed platelets when triggered bythrombin or collagen. EPA intake failed to affect the oxygenated metabolism of AA, measured by the formation of TXB2, HHT and 12-HETE from either exogenous or endogenous (thrombin stimulation) AA. Formation of AA oxygenated products including 6-keto-PGFla in clotted blood was unaffected. Excretion of urinary TXB2, 6-keto-PGFlα and their 2,3-dinor metabolites wasleft unchanged by ingestion of EPA. Platelet, but not plasma α- and γ-tocopherols were significantly increased by EPA intake. We conclude that, in our experiment, the reduction of platelet aggregation could not be explained by modification of AA metabolism. Since vitamin E has been associated with a decrease ofplatelet aggregation, at least in vitamin E deficiency, and platelet vitamin E being reduced with age, we speculate that the decreased platelet aggregation after EPA intake might be related to the increased platelet tocopherols.
ENHANCED THROMBIN-INDUCED AGGREGATION AND INOSITOL TRISPHOSPHATE FORMATION OF PLATELETS FROM SPONTANEOUSLY HYPERCHOLESTEROLEMIC RATS
Authors: Winocour P P , Rand P D , Vickers J D , Kinlough-Rathbone R L , Mustard J F
Published: August 2018

Platelets from rats with diet-induced hypercholesterolemia are hypersensitive to thrombin through a pathway independent of released ADP or thromboxane A2 (TXA2) formation. We examined if platelets from rats with spontaneous hypercholesterolemia (HC) are similarly hypersensitive. HC rats (plasma cholesterol: 130±4 mg/dl, n=15) were compared with their normocholesterolemic genetic controls (NC) (87±4 mg/dl, p<0.001, n=16). Total cholesterol/109 platelets was not different between the groups (HC: 0.314±0.032 μmole, n=7; NC: 0.357±0.046 ymole, n=7). Washed platelets were prelabelled with 14c-serotonin. In the presence of aspirin (to inhibit TXA2 formation) and creatine phosphate/creatine phosphokinase (CP/CPK) (to remove released ADP), HC platelets aggregated more (22±2%, n=ll) than NC platelets (10±4%, n=12, p<0.01) in response to thrombin (0.065 U/ml); 14C release was not different. Thrombin causes inositol mono-, bis-, and trisphosphate (IP, IP2, IP3) formation from phosphoinositides (PI, PIP, PIP2 respectively) in rabbit platelets; IP3 may be involved in Ca++ mobilization and the release of granule contents. We examined if enhanced inositol phosphate formation was associated with hypersensitivity of HC platelets. Platelets were prelabelled with 3H-inositol and stimulated with thrombin (0.057 U/ml) for 30 sec in the presence of aspirin and CP/CPK. Li+ (20 mM) was used to prevent degradation of inositol phosphates to inositol. 3H-IP, IP2 and IP3 were isolated by ion-exchange chromatography. The increase in radioactivity (dpm/109 platelets) in IP2 and IP3 following thrombin stimulation was greater in HC platelets (IP2: 2210±160, n=4; IP3: 1430±180, n=4) than in NC platelets (IP2: 660±150, n=4, p<0.001; IP3: 490±100, n=4, p<0.01); IP was not different.

Thus platelets from spontaneously HC rats are hypersensitive to thrombin independently of released ADP or TXA2 formation. This hypersensitivity is associated with only moderate increases in plasma cholesterol and no detectable increase in total platelet cholesterol. Enhanced labelling of IP3 may indicate that enhanced activity of the pathways leading to IP3 formation is associated with this hypersensitivity.
IMMUNOLOGICAL ASSAYS FOR THE Ca(II)-DEPENDENT AND NONCa(II)-DEPENDENT CONFORMATIONS OFJtLUMAN PROTELN Z (PZ)
Authors: van der Meer F J M , van Tilburg N H , Poort S R , Briët E , Bertina R M
Published: August 2018

Human PZ was purified using bariumcitrate absorption and elution, DEAE-Sephadex chromatography and gelfiltration on Sephadex G-200. The purified protein was used for the development of a specific rabbit antiserum, which could be used in a electroimmunoassay (EIA). Using immunoaffinity procedures, two populations of antibodies were isolated from the antiserum: one against the Ca(II)-dependent conformation of PZ(Ca(II)PZAg) and one against the nonCa(II)-dependent conformation (nonCa(II)PZAg). These antibody populations were used for the development of specific and highly sensitive immunoradiometric assays for Ca(II)PZAg and nonCa(II)PZAg. In healthy volunteers PZ levels varied from 0.28 to 1.96 U/ml (mean value 0.99 ± 0.34 U/ml). The observed variation was much more extensive than for other vitamin K-dependent proteins. Repeatedly testing of individuals with low PZAg levels gave comparable results, excluding acute phase reactivity of PZ. The mean value for the Ca(II)PZAg/nonCa(II)PZAg ratio was 1.04 ± 0.27 (range 0.51 - 1.58).

Interestingly, PZ antigen levels were found to be severely reduced in 60 plasmas of patients stably treated with oral anticoagulants. At increasing intensity of treatment nonCa(II)PZAg decreased: 0.14 ± 0.04 (INR 2.6), 0.12 ± 0.04 (INR 3.0), 0.09 ± 0.03 (INR 3.4), 0.08 yp 0.03 (INR 3.8), 0.06 ± 0.01 (INR 4.2) and 0.06 ± 0.02 U/ml (INR >4.4).

The decrease in nonCa(II)PzAg was much more extensive than observed for the nonCa(II)Ag of other vitamin K dependent factors (factors II, VII, IX, X, protein C and protein S).

In general concentrations of Ca(11)PZAg were more reduced than those of nonCa(II)PZAg (ratio: ∼ 0.7). This ratio was virtually independent of the intensity of oral anticoagulant treatment.
USE OF A MONOCLONAL ANTIBODY TO MEASURE THE SURFACE EXPRESSION OF THROMBOSPONDIN FOLLOWING PLATELET ACTIVATION
Authors: Legrand C , Dubernard V , Kieffer N , Nurden A T
Published: August 2018

A radiolabelled monoclonal antibody (mAb) against native thrombospondin (TSP) has been used to quantitatively assess the surface exposure of intracellular TSP following platelet stimulation. This mAb, designated 5G11, was purified from ascitic fluid by ammonium sulfate precipitation followed by chromatogloghy on DEAE Trisacryl. The isolated IgG were labelled with I by the chloramine T method (sp.act. 200-500 cpm/ ng). The specificity of the mAb was established by immunoblot-ting and crossed immunoelectrophoresis using platelet protein extracts. When the labelled IgG (20 μg/ml) were incubated with resting platelets in Tyrode's buffer binding was of the order of 2,000 molecules per platelet. Binding was increased 2 fold and 5-7 fold respectively upon ADP- and thrombin-(or ionophore A23187) stimulation. Unactivated platelets from 2 patients with the Gray Platelet Syndrome bound baseline levels of 5G11, but binding did not increase after platelet activation. In the presence of saturating concentrations of mAb 5G11, an average of 30,000 molecules of IgG were bound by normal platelets stimulated by thrombin. This binding was strongly reduced in the presence of EDTA. It was not significantly affected by AP-2, an anti-GP IIb-IIIa monoclonal antibody which inhibited by more than 85% the binding of plasma fibrinogen but which did not inhibit the surface expression of platelet fibrinogen. It was decreased but not prevented by the presence of an excess of rabbit anti-fibrinogen Fab fragments during the stimulation, while binding at the lower end of the normal range was observed on two different occasions using platelets isolated from an afibrinogenemic patient lacking platelet fibrinogen. These results suggest that while platelet fibrinogen may contribute to the surface organization of TSP other component(s) are required for the full expression of TSP on the platelet surface.
THROMBOSPONDIN SPECIFICALLY INTERACTS WITH AMINO ACID SEQUENCES WITHIN THE A α- AND B β- CHAINS OF FIBRINOGEN
Authors: Bacon-Baguley Theresa , Kendra-Franczak Suzanne , Walz Daniel
Published: August 2018

Thrombospondin (TSP) is responsible for the secretion-dependent phase of platelet aggregation. The mechanism of this action is believed to be through the binding of TSP to fibrinogen, resulting in the stabilization of the platelet aggregate. It has been established that the binding of fibrinogen to the platelet surface is dependent upon peptide sequences present, respectively, in the Aa- and y-chains. We have hypothesized that the binding of TSP to fibrinogen is also dependent upon unique fibrinogen peptide sequences. To test this hypothesis we have examined the interaction of TSP and f.ih.r.inogen. using..a.-blat-b.inding assaLy of reduced fibrinogen, the separated fibrinogen chains, selected fibrinogen domains or peptides generated from cyanogen bromide cleaved chains. Iodinated TSP bound specifically to the Aα - and Bβ - chains. Binding to these chains was calcium independent, mutually exclusive and could be blocked either by preincubation of TSP with 9.4 μ M fibrinogen or by preincubation of fibrinogen with 1.1 nM thrombospondin. TSP bound to the D and DD plasmin fragment of fibrinogen; TSP interacted exclusively with the B-chain component of the DD fragment. The cyanogen bromide fragments of the separated Aα - and Bβ -chains were resolved through a combination of gel filtration and reverse-phase chromatography. TSP was found to bind to a single peptide within these fibrinogen chains. These studies demonstrate that thrombospondin interacts with at least two distinct sites on fibrinogen, and these sites differ from those already described for fibrinogen binding to platelets.
THROMBOSPONDIN INTERACTION WITH PLASMINOGEN
Authors: DePoli Patricia , Bacon-Baquley Theresa , Walz Daniel A
Published: August 2018

Platelet thrombospondin (TSP) interacts with plasminogen in a specific and saturable manner. TSP can form a trimolecular complex with histidine-rich glycoprotein and plasminogen and the plasminogen within such complexes can reportedly be activated by tissue plasminogen activator. We have studied the interaction of TSP with plasminogen using Western blotting of plasminogen, reduced plasmin and the elastase-generated fragments of plasminogen and their binding of iodinated TSP. TSP was found to specifically bind to plasminogen and the heavy (non-enzyme) chain of plasmin in a calcium-independent manner. Binding could be blocked by preincubation of the immobilized plasminogen or plasmin with an excess of unlabeled TSP. Plasminogen domains (kringles) were generated by limited eTastase proteolysis. TSP bound specifically to a single 51 kDa plasminogen fragment. The elastase-generated fragments were separated by lysine-Sepharose chromatography and their identities established by amino acid composition and amino-terminal sequence analysis. The 51 kDa plasminogen fragment bound to lysine-Sepharose and had an amino-terminal sequence corresponding to kringle 4 (K4) and a composition consistent with that of K4-K5-plasmin. TSP binding to this fragment was not blocked by the presence of an excess of the fragment K1-K2-K3, K4, nor miniplasminogen (K5-plasmin). Binding does not appear to be directly dependent upon the specific high-affinity lysine binding site of the 51 kDa fragment. Our data suggests that thrombospondin interacts with plasminogen at a single distinct site, and that this recognition site is at or near the K4-K5 contiguous region of plasminogen.
MOLECULAR MARKERS OF HEMOSTATIC ACTIVATION: COMPARISON OF PLASMA AND URINARY LEVELS
Authors: Ashley H W , Fareed J
Published: August 2018

We have compared plasma and urine levels for both normal control and aspirin treated groups (n=ll) using standard methods for fibrinopeptide, A (FPA), platelet factor 4 (PF-4), g-throm-boglobulin (β-TG), Bβ; 15-42 peptide (BβP), and tissue plasminogen activator (t-PA). Normal males and females of varying ages (22-50) made up the two groups. For aspirin treatment, 300 mgs/ day of aspirin was given PO for 3 days prior to the 24-hour urine and blood collection. The following data was generated:

These results indicate that aspirin treatment produces a marked decrease of both plasma and urinary levels of molecular markers of hemostatic activation, suggesting that aspirin produces its effect at multiple sites within the hemostatic network. Profiling of urinary markers of hemostatic activation may also prove useful in the evaluation of prothrombotic states and evaluation of antithrombotic drugs.
PROGNOSTIC SIGNIFICANCE OF HAEMOSTATIC VARIABLES IN ACUTE STROKE
Authors: Douglas Jessie T , Lowe G D O , Balendra R , Forbes C D , Creighton L J , Gaffney P J , Lane D A , Kluft C
Published: August 2018

Acute stroke is normally the result of thromboembolism. Such thromboemboli form and extend by the interaction of platelets and fibrin and elicit a fibrinolytic response. Hence laboratory indices of platelet activation, thrombin formation and plasmin formation may be related to thrombus size and progression, and hence to clinical outcome, ie disability and death. We studied 100 patients with acute paretic stroke and followed them for 1 year. Plasma levels of betathromboglobulin (BTG), fibrinogen, fibrinopeptide A (FPA), fibrin(ogen) fragment BB15-42, serum fragment E, high molecular weight cross-linked fibrin degradation products (X-L FDP) D-dimer, total and tissue plasminogen activator activity, tissue plasminogen activator inhibition and serum fibrin(ogen) degradation products (FDP) were related to death and functional recovery in the 1 year follow up period. The levels of BTG, fibrinogen, FPA, BB15-42, tissue plasminogen activator inhibition and serum fragment E were significantly higher on the first day following stroke, in patients who subsequently died within 1 year when compared to patients who survived. Lowered levels of fibrin plate lysis area as well as raised tPA activity, X-L FDP and D-dimer levels did not achieve significance in patients who subsequently died. In the patients who survived only increased BB15-42 and X-L FDP levels were predictive of functional dependence when compared to patients who became independent. Increased fibrinogen, FPA and fibrin plate lysis area, fragment E and tPA activity and reduced D-dimer did not achieve significance in patients with greater disability when compared to patients with minimal disability. We conclude that several measures of activation of haemostasis are predictive of death in the year following stroke, but only BB15-42 and X-L FDP predict disability in survivors.
DETECTION OF FACTOR X ACTIVATION IN HUMANS
Authors: Bauer K A , Kass B L , Bednarek M , Kloczewiak M , Hawiger J , Rosenberg R D
Published: August 2018

The activation of factor X by factor VII/VIIa-tissue factor or factor IXa plays a pivotal role in the hemostatic mechanism. This reaction results in the liberation of a peptide from the zymogen for which we have developed a sensitive and specific radioimmunoassay (RIA), The native peptide was purified from activated human factor X by hydroxylapatite chromatography and reverse-phase high pressure liquid chromatography (HPLC). Gel filtration experiments demonstrated that the peptide was not physically associated with the enzyme. A 15 amino acid peptide with the COOH-terminal sequence of the activation fragment was synthesized using the solid-phase method of Merrifield. Antisera were raised in rabbits to the synthetic analogue coupled to bovine serum albumin with glutaraldehyde. The antibody population obtained was used to construct a double antibody RIA and was able to measure as little as 0.02 nM of this component. The antibody reactivity toward the factor X zymogen was negligible (less than 1/36,000 that of the activation peptide on a molar basis). However because other plasma constituents contributed to a nonspecific basal signal in the RIA, we developed an extraction procedure for the native peptide utilizing perchloric acid. Plasma peptide levels in normal individuals were ∼0.1 nM, and elevations up to 0.8 nM were observed in patients with evidence of disseminated intravascular coagulation. Individuals chronically anticoagulated with coumarin derivatives had plasma levels of this peptide suppressed to ∼0.02 nM. The validity of our measurements of factor X activation in vivo is supported by the fact that the immunoreactive signal migrates on reverse-phase HPLC in a manner identical to that of the native activation peptide and can be quantitatively recovered. This assay should be useful for studying the pathophysiology of thrombotic as well as bleeding disorders in humans.
CHARACTERIZATION OF RECOMBINANT HUMAN TISSUE-TYPE PLASMINOGEN ACTIVATOR MISSING THE FINGER DOMAIN
Authors: Higgins Deborah L , Holmes William E
Published: August 2018

Site-specific mutagenesis was used to produce a mutant form of tissue-type plasminogen activator (t-PA) which was missing the first 44 amino acids. This domain has sequence homology with the type 1 regions in proteins such as fibronectin and is commonly called the finger domain. The mutant protein (des 1-44 t-PA) was expressed in Chinese Hampster Ovary cells, and was purified using chromatography on Zn-chelate sepharose and lysine-sepharose. Sequence analysis indicated that the resulting protein was homogeneous and started at amino acid 45 in the sequence of the normal protein. The two-chain forms of both des 1-44 t-PA and normal sequence t-PA exhibited similar kinetic constants with a small synthetic substrate (H-D-Isoleucyl-L- prolyl-L-arginyl-p-nitroani 1 ide). The ability of des 1-44 t-PA to activate plasminogen was decreased to 70% of the rate of normal t-PA. The rate of plasminogen activation by normal t-PA was stimulated 51-fold in the presence of fibrin, whereas with des 1-44 t-PA it was stimulated only 40-fold. Although des 1-44 t-PA bound to lysine-agarose, little (if any) binding was observed to either intact or degraded fibrin indicating that fibrin stimulation is due in part to the ability of t-PA to recognize plasminogen bound to fibrin as a preferable substrate. The mutant t-PA was capable of forming complexes in vitro with all of the inhibitors in blood which react with normal sequence t-PA. The rate of reaction with α2-macroglobulin, however, was slower with des 1-44 t-PA than with normal sequence t-PA. The similar resistance of des 1-44 t-PA and normal sequence t-PA to proteolysis and the ability to react with a battery of monoclonal antibodies suggests that the deletion did not cause perturbed folding, but rather that alterations in function of des 1-44 t-PA were due to the lack of the finger domain.
ROLE OF PROTEOLYSIS AT ARGININE-275 OF TISSUE PLASMINOGEN ACTIVATOR (t-PA) AS ASSESSED BY SITE-DIRECTED MUTAGENESIS
Authors: Vehar G A , Tate K M , Higgins D L , Holmes W E , Heyneker H L
Published: August 2018

The significance of the cleavage at arginine-275 of human t-PA has been the subject of debate. It has been reported, as expected for a member of the serine protease family, that the single chain form is a zymogen and that generation of catalytic activity is dependent upon cleavage at arginine-275. Other groups, in contrast, have found considerable enzyme activity associated with the one-chain form of t-PA. To clarify the functional significance of this proteolysis and circumvent cleavage of one-chain t-PA by itself or plasmin, site-directed mutagenesis was employed to change the codon of arginine-275 to specify a glutamic acid. The resulting plasmid was used to transfect CHO cells. The single chain mutant [Glu-275 t-PA] was expressed in CHO cells and the protein purified by conventional techniques. The mutant enzyme could be converted to the two-chain form by V8 protease, but not by plasmin. Glu-275 t-PA was 8 times less active in the cleavage of a tripeptide substrate and 20-50 times less active in the activation of plasminogen in the absence of firbrin(ogen) than its two-chain form. In the presence of fibrin(ogen), in contrast, the one and two-chain forms of Glu-275 t-PA were equal in their ability to activate plasminogen in the presence of fibrin(ogen). The activity in these assays was equal to the activity of wild type t-PA. In addition, it was observed that fibrin bound considerably more of the one-chain form of t-PA than the two chain forms of t-PA and the Glu-275 mutant. The one and two-chain forms of the wild type and mutated t-PA were found to slowly form complexes with plasma protease inhibitors in vitro, although the one-chain forms were less reactive with alpha-2-macroglobulin. It can be concluded that the one-chain form of t-PA appears to be fully functional under physiologic conditions and has an increased affinity for fibrin compared to two-chain t-PA.
IN VITRO CHARACTERIZATION OF THE PROCQAGULANT ACTIVITY WITHIN THE BRONCHQALVEOLAR COMPARTMENT OF NORMAL HUMAN LUNG
Authors: Fair D S , Chapman H A , Allen C L , Yee R
Published: August 2018

The procoagulant activity (PCA) of the normal human broncho-alveolar (BA) lining layer was examined qualitatively and quantitatively. Unconcentrated, cell-free lavage freshly obtained from normal volunteers clotted whole plasma in 84 ± 20 sec. The PCA was initiated by factor VII (F VII) and tissue factor (TF) complexes as judged by differential activities in various plasmas genetically deficient in single clotting factors and by neutralization of the PCA with antibodies to either F VII or TF. The cell-free fluid contained about 8500 thromboplastin units/mg total protein. The fluid also contained F VII activity estimated by clotting activity to be 2 ng of plasma F VII/ml of lavage fluid or 22 ng/mg total protein compared to 7 ng/mg protein for normal human plasma. Amidolytic activity measurements suggested that the F VTI concentration was 0.57 ng/ml of lavage fluid. Because of the increased amounts of F VII expressed in the lung relative to plasma, we investigated the activation state of the F VII by the ratio of clotting to amidolytic (VIIc/VIIam) activities. The ratio of unfractionated alveolar fluid was about 19, suggesting the presence of the more active two-chain F Vila. However, imnunoblots of concentrated lavage protein revealed only single-chain F VII and 125I-F VII added to the fluid was not converted to 125I-F VIIa. Additional studies showed that the enhanced PCA of lavage fluid F VII over that of plasma was due to a factor (s) in the sedimentable fraction of the lining layer which directly increased the prothrcmbinase activity of exogenous, purified factor Xa and V/Va and phospholipid (PL). Although normal alveolar macrophages express TF and F VTI, we estimate that the cells contribute < 15% of the total PCA within the alveolar compartment. However, these cells are the only suitable PL surface for prothrcmbinase complex formation within the lavage material, suggesting that macrophages are a major site for thrombin formation within alveoli. Thus, it appears the enhanced rates of thrombin formation rather than an increase in the rate of factor Xa generation explain the accelerated clotting times and the high F VIIc/VIIam ratio, and that macrophages may play a significant role in the total PCA output observed in the lung.
HEPARIN BINDING TO HUMAN MONOCYTES: MODULATION BY HISTIDINE-RICH GLYCOPROTEIN
Authors: Leung L , Saigo K , Grant D
Published: August 2018

Heparin and its related glycosaminoglycans interact with a variety of cell types and, irrespective of their anticoagulant activities, have a complex and biologically important influence in blood vessel wall biology. In this study, the binding of heparin to the human monocytoid cell line U937 was characterized. 35s-Heparin bound to U937 cells in a specific, concentration-dependent, and saturable manner, while binding to human erythrocytes was minimal. The binding was rapid, reaching a steady state at 30 min at 4°C and was reversible. Excess unlabeled heparin, but not chondroitin sulfate or dermatan sulfate, inhibited heparin binding, demonstrating the specificity for heparin. Scatchard plot analysis revealed a single class of heparin binding sites, with an apparent Kd of 0.32 uM and approx. 2.0 × 106 sites/cell. When binding was performed at 37°C, significant amount of cell-bound heparin was not removed by cell surface trypsinization, indicating endocytosis of heparin by U937 cells. Taken together the binding characteristics suggested the presence of specific heparin receptors on U937 cell surface. Histidine-rich glycoprotein (HRGP), a potent heparin-binding protein found in human plasma and platelets, decreased the affinity of heparin for U937 cells without a significant change in maximal binding. This negative modulation of heparin binding was specific for HRGP since antithrombin III was much less effective. While EDTA abolished the HRGP-heparin interaction, it had no effect on the HRGP modulation of heparin cell binding, suggesting that HRGP interacted directly with the U937 cell and altered its affinity for heparin. Specific binding of labeled HRGP to U937 cells was demonstrated. Cell-surface bound heparin markedly accelerated the inactivation of thrombin by antithrombin III. Its anticoagulant potency was equivalent to heparin in the fluid phase. Human monocytes and macrophages provide an optimal cell surface for the assembly of prothrombinase and the generation of thrombin. The specific binding of anticoagulantly active heparin to monocytes and macrophages may modulate the procoagulant properties of these cells at sites of inflammation and thrombosis. Furthermore, since heparin fractions with high or low affinity for antithrombin III bound equally well to U937 cells, heparin may have a direct effect on monocyte cell biology. The current study also raises the possibility that the significant modulation of heparin cell binding by HRGP may represent a major function for this protein in vivo.
FIBRINOGEN BINDS TO HUMAN NEUTROPHILS AT A SITE DISTINCT FROM GPIIb/IIIa
Authors: Gustafson E J , Lukasiewicz H , Schmaier A H , Niewiarowski S , Colman R W
Published: August 2018

Many observations suggest a potential role for neutrophils in the modulation of hemostasis and thrombosis. Arterial thrombi are characterized by the presence of large numbers of neutrophils lining the perimeter of platelet aggregates. While investigating binding of high molecular weight kininogen (HMWK) to neutrophils, we found that fibrinogen (Fb) could inhibit binding of 125I-HMWK as well as displace HMWK already bound to neutrophils. We therefore initiated studies to determine whether Fb could bind to human neutrophils. Both Zn++ and Ca++ were required for maximal binding of 125I-Fb to neutrophils. Binding did not occur with Ca++ (ZmM) alone and was only 1/3 the maximal amount with Zn++ (50 μM) alone. At 4° the amount of 125I-Fb bound to neutrophils reached a plateau by 15 minutes and remained at this level over the next 30 minutes. At 23° and 37° the amount of 125I-Fb bound peaked by 4 minutes and then decreased over the next 30 minutes indicating receptor-mediated internalization. Excess Fb inhibited binding of 125I-Fb to neutrophils while prekal1ikrein, factor XII, and fibronectin did not. Binding of 125I-Fb was 99% reversible at 4° within 10 minutes with a 50-fold molar excess of Fb and 90% displaceable by excess HMWK. The apparent Kd was approximately 0.45 μM. Arg-Gly-Asp-Ser (RGDS) is a tetrapeptide common to Fb, fibronectin, vitronectin and other cel 1-attachment proteins. Fb has been demonstrated to bind to the glycoprotein IIb/111 a (GPIIb/IIIa) complex which is the platelet membrane receptor for RGDS. Although this RGDS-GPIIb/IIIa interaction occurs with Fb binding to platelets, it is apparently not involved with Fb binding to monocytes. To investigate if Fb binding to neutrophils involved this interaction of GPIIb/II la -RGDS we performed further studies. Binding of 125I-Fb to neutrophils was not inhibited by RGDS nor was it inhibited by a monoclonal antibody (10E5) to the platelet GPI I b/IIIa complex. In addition, the amount of 125I-Fb that hound to neutrophils from a patient with Glanzman's thrombosthenia was the same as that bound to normal neutrophils. These studies indicate that human neutrophils specifically bind Fb at a site similar to HMWK and distinct from GPIIb/IIIa.
HIGH MOLECULAR WEIGHT KININOGEN SPECIFICALLY BLOCKS THROMBIN-INDUCED AGGREGATION BY INHIBITING PLATELET CALPAIN
Authors: Puri R N , Zhou F , Bradford H , Gustafson E J , Colman R F , Colman R W
Published: August 2018

We have previously shown that platelet-aggregation induced by alpha-thrombin (1.7 nM) involves complete cleavage of the surface membrane polypeptide, Mr = 100 kDa (MP 100) labeled by FSBA in intact platelets. The failure to cleave MP100 in membrane preparations or in platelets treated with metabolic inhibitors or leupeptin, suggested that thrombin was acting by activating platelet calpain. Since high molecular weight kininogen (HMWK) is the most potent plasma inhibitor of calpain(s), we now report that HMWK inhibited thrombin-induced aggregation in a dose-dependent manner over a range of plasma concentrations. HMWK did not inhibit aggregation induced by ADP, collagen, U46619, A23187 and/or PMA. In order to study the action of HMWK in a plasma environment we utilized Y-thrombin. The aggregation induced by γ-thrombin (25 nM) in washed human platelets was also inhibited by HMWK. A much higher concentration of γ - thrombin (200 nM) was required to induce similar aggregation of platelets suspended in normal plasma. In contrast, γ -thrombin (50nM) induced complete aggregation of platelets suspended in plasma completely deficient in total kininogen indicating that a kininogen was predominently responsible for the inhibitory effect of plasma. When platelets were suspended in plasma deficient only in HMWK, aggregation required 75 nMY-thrombin. When plasma deficient in HMWK was supplemented with a physiological concentration of HMWK (0.67 μM) the aggregation of suspended washed platelets was similar to that in normal plasma. Finally, we found that purified platelet calpain-2 not only exposed fibrinogen binding sites and induced platelet aggregation, but also completely cleaved MP 100 in both intact platelets and membrane preparations. We conclude: a) Thrombin-induced platelet aggregation involves the indirect proteolytic cleavage of MP100 by activating calpain-2, and b) Inhibition of thrombin-induced platelet aggregation by HMWK involves specific inactivation of platelet calpain.
DISSOCIATION BY EDTA OF THE HUMAN PLATELET GP IIb-IIIa COMPLEX PREVENTS ADP-INDUCED FIBRINOGEN DEPENDENT AGGREGATION BUT NOT THROMBIN-INDUCED THROMBOSPONDIN DEPENDENT AGGREGATION
Authors: Cazenave J-P , Bay-Sutter A , Gachet C , Lanza F , Stierlé A
Published: August 2018

The aggregation of washed human platelets by ADP does not cause the release reaction and is dependent, following ADP binding to its receptor, on exposure of the membrane glycoprotein (GP) IIb-IIIa complex, platelet contact and presence in the external medium of Ca2+ and fibrinogen. EDTA, which chelates Ca2+ can inhibit platelet aggregation by removal of Ca2+ from external medium or by dissociating the GPIIb-IIIa calcium dependent complex. We have studied the effects of prolonged incubation of human platelets with EDTA on the platelet ADP-fibrinogen dependent aggregation. Human platelets were washed according to a modification of Mustard’s method (Ann. Biol. Clin. 1983, 41, 167-179). Platelets in the second washing fluid were labeled with 3H-5HT and split in 2 parts : one part (A) was incubated with 5 mM EDTA for 30 min at 37 °C, the other (B) with Tyrode buffer. A and B were centrifuged and the platelet pellet resuspended in Tyrode-0.35 % albumin buffer with apyrase at 37°G at 300,000 platelets per mm3. In contrast to control platelets (B), EDTA-treated platelets (A) : i) did not aggregate to ADP (1-100 uM) in the presence of added human fibrinogen, ii) did not bind 125I-fibrinogen, iii) had a dissociated GPIIb-IIIa on crossed immunoelectrophoresis of 125I-labeled platelets (lactoperoxydase method) after migration against a rabbit anti-human platelet antibody, iv) did not aggregate to ZnCl2 or monoclonal antibody (Mab) 609. In response to thrombin (0.01-0.5 U/ml), EDTA-treated platelets (A) released 3H-5HT and aggregated but to a lesser extent than B platelets. Thrombin induced aggregation of EDTA-treated platelets'was inhibited by 5 mM EDTA or anti-TSP antibody or by 0KM5 (Ortho), a Mabagainst the TSP receptor. In conclusion, dissociation of GPIIb-IIIa of human platelets by EDTA inhibits completely the aggregation depending on fibrinogen, but platelets can aggregate when they are stimulated by thrombin through release of TSP which binds to its receptor.
DETECTION OF INACTIVATED α2-MACROGLOBULIN (α2M) IN HUMAN PLASMA USING MONOCLONAL ANTIBODIES
Authors: Abbink J , Nuijens J , Huijbregts C , Hack E
Published: August 2018

Monoclonal antibodies (mAbs) were raised against human a2M. Five mAbs that bound to α2M in ELISA were further analyzed by a radioimmunoassay (RIA) for their reaction with three types of α2M: native α2M, chemically inactivated α2M (iα2M) (methylamine treated), and proteolytically iα2M. One mAb reacted with all forms of α2M, while four mAbs bound both forms of ia2M but not native α2M. One of these latter mAbs (Ml) was used to develop a RIA (the Ml-assay) for the detection of iα2M in plasma: Ml coupled to Sepharose is incubated with the plasma to be tested, and bound iα2M is detected by a subsequent incubation with polyclonal 125I-anti-α2M antibodies. As little as 5 ng of iα2M can be detected with this assay in the presence of an excess of native α2M. This assay was then applied to measure inactivation of α2M in vitro and in vivo. In vitro activation of the contact system in plasma by dextran sulfate results in the inactivation of ca 10% of α2M. When blood from normal donors was collected under optimal conditions, about 0.5% of the total α2M content appeared to be iα2M. Longitudinal studies in patients (a.o. with septicaemie, during cardiopulmunary bypass) revealed that increased levels of iα2M occurred sporadically. The Ml-assay appears to be useful to monitor the role of α2M in human diseases.
MOLECULAR HETEROGENEITY IN FAMILIAL HEPARIN COFACTOR II DEFICIENCY
Authors: Andersson T R , Larsen M L , Abildgaard U
Published: August 2018

In our normal material of 379 blood donors,3 indivi-uals had values below mean −2.5 SD (below 56%).Further studies revealed hereditary deficiency in two of these individuals. In the family study,5 out of 7 individuals had heparin cofactor II (HC II) values below 56%. In only one of these 5 a history of DVT was obtained. In deceased members of her family, however, frequent episodes of thromboembolic disease had occurred.

Crossed immunoelectrophoresis (CIE) was performed in plasma from deficiency individuals from both families. Heparin in the first dimension gave a pattern similar to that observed with normal pooled plasma. The inactivation of thrombin by HC II is preferentially accelerated by dermatan sulfate (DS).HC II consumption in in vitro coagulation is increased by DS rather than by heparin. This prompted the addition ofTBS to the first dimension, which makes the antigen move faster, but produced no alteration in the antigenic pattern or size, neither in pooled plasma nor in a family member with normal activity. In family members with low HC II activity, DS in the first dimension, resulted in an abnormal CIE pattern with two distinct precipitation arcs with identity pattern. Molecular heterogeneity of HC II has previously not been reported.

Screening 70 individuals who had sustained thrombosis before the age of 50,no values suggesting congenital deficiency were encountered.

Conclusion. The CIE findings suggest that plasma of affected family members contain two types of HC II molecules of immunological identity. One type shows the normal accelerated mobility in the presence of DS and the other lacks this effect. The abnormal electrophoretic pattern becomes, however, first apparent when DS is added to the first dimension. When looking for molecular defects in congenital HC II deficiency, it is important that the first dimension is run with DS.
PROMOTOR MUTATIONS IN A PATIENT WITH HAEMOPHILIA B LEYDEN
Authors: Reitsma P H , Riemens A M , Bertina R M , Briít E
Published: August 2018

Haemophilia B Leyden is characterized by low levels of factor IX antigen and activity before the age of 15, whereas after puberty factor IX levels rise at a rate of about 4-5% per year. To date such a genetic variant of factor IX synthesis has been reported in two (probably related) Dutch families, in a Greek and in an American family of Armenian descent. Laboratory and clinical investigations indicate that the factor IX protein is normal but that the regulation of factor IX synthesis has come under the control of the steroid hormone testosterone. We have started to investigate the factor IX gene in a patient from a Dutch family in an attempt to explain the aberrant regulation.

Southern blotting revealed no gross deletions or insertions in the factor IX gene. Therefore the promotor region of the factor IX gene was cloned and subjected to a detailed restriction enzyme analysis. This also did not indicate that significant DNA deletions or insertions had occurred. Subsequently we established the nucleotide sequence of the DNA surrounding the first exon which encompassed about 600 basepairs of the promotor region. Two deviations from previously published sequence data were recorded. Firstly, an A T change was noted in the presumed "tata" box region 20 basepairs upstream from the start site of mRNA synthesis. Secondly, at position −423 a T C change was found which lies 13 basepairs upstream from a potential alternative "tata" box.

The point mutation at position −20 might well explain the failure of gene expression during the prepuberal stage of the disease. Whether the same point mutation also leads to the testosterone effects or that a second sequence variation is a prerequisite for this phenomenon remains to be established from studies on the promotor region in representatives of the Greek and American families. Eventually the introduction of chaemeric genes, containing the various promotor regions, into testosterone responsive cells should delineate the promotor sequences responsible for the variations in factor IX gene expression.
MOLECULAR ANALYSIS OF COAGULATION FACTOR VIII AND IX GENES BY DNA PROBES
Authors: de la Salle C , Baas M J , Grunebaum L , Gialeraki R , Mandalaki T , Cazenave J-P
Published: August 2018

About 250 individuals belonging to 44 families with hemophilia A or B were studied in our laboratory. The detection of carriers was first established by pedigree analysis of each family . and coagulation and immunological assays of factor VIII or IX. The availability of specific probes for the molecular study of these two genes makes possible a diagnosis with certainty in the case of informative families. 25 families of hemophilia A were studied. For each person, blood was collected into EDTA and leucocyte DNA was extracted, digested by restriction endonucleases, electrophoresed in 0.9 % agarose gels and transferred to nitrocellulose filters by Southern blotting. Two probes were used for the analysis of factor VIII gene. The St 14 probe (J.L. Mandel) located on the q28 region of the X chromosome and closely linked to the gene, determines a restriction fragment length polymorphism (RFLP) when the DNA is digested by the enzyme TaqI. The p114-12 genomic probe (Genentech) corresponding to the exons 17 and 18 of the factor VIII gene, reveals a RFLP in the DNA digested by the enzyme BclI. 19 families -of hemophilia B were studied. A total factor IX cDNA probe was used for the screening of potential deletions in the case of hemophiliacs with circulating antibodies. A genomic probe containing the exons II, III and IV of factor IX was used to detect the TaqI RFLP. For the study of factor VIII gene, the extragenic probe St 14 gives a very high percentage of informativity (about 90 %) but recombination can occur between the probe and the gene. The p 114-12 probe, which is used to confirm the results given by the St 14 probe, gives about 20 % informativity. In our study, we were able to diagnose carrier state with certainty in 92 % of the families. For hemophilia B, the genomic probe gives about 40 % informativity. A large deletion of the region of the factor IX gene has been found in one family and remains to be mapped. In conclusion, carrier detection and prenatal diagnosis can be established with certainty by molecular studies in most cases of hemophilia A using the St 14 probe, with a 5 % risk of recombination when the BclI RFLP cannot confirm. This diagnosis is possible in about 40 % of the cases of hemophilia B.
FACTOR V IS ACTIVATED AND CLEAVED BY PLATELET CALPAIN: COMPARISON WITH THROMBIN PROTEOLYSIS
Authors: Colman Robert W , Bradford Harlan , Annamalai Anjanayaki
Published: August 2018

Platelets are known to process human and bovine factor V during secretion and/or membrane binding. We therefore studied the functional and structural changes produced in human factor V and Va by purified platelet calpain. A maximum increase in factor V coagulant activity of 2.5-fold over control incubations was observed for calpain (0.6 u/ml) at 25°C in comparison with a 10-fold increment for a thrombin (1 u/ml). Thrombin addition to reactions initiated by calpain resulted in further activation comparable to that of thrombin alone, while subsequent addition of calpain had no effect on the extent or pattern of the activation of factor V by thrombin. The cleavage pattern of factor V produced by these two enzymes are distinctly different. Calpain yields initial components of 210 kDa and 160 kDa within 1 min. Further digestion of the 210 kDa species give rise to polypeptides of 150, 140 and 120 kDa by 2 min with and increase in coagulant activity. The degradation of the 160 kDa polypeptide gives rise to smaller fragments of 130, 100, 90, and 87 kDa. Immunob lot ting of these fragments with the monoclonal antibody B10 directed to factor V and the thrombin generated Cl fragments yields results demonstrating an immunological relationship to the calpain generated components of 210, 160, 140 and 120 kDa. Thus platelet calpain generates a complex cleavage pattern different from thrombin which may explain the partial activation observed.