bluuebunny/crossref_metadata_2025 huggingface dataset:
THE FUNCTIONAL DOMAINS OF COAGULATION FACTOR VIII
Authors: Rosenberg Steven , Hartog Karin , Nordfand Ole , Ezban Mirella , Lyn Burke Rae
Published: August 2018

The functional domains of Factor VIII have been investigated using site-directed mutagenesis to probe the effect of thrombin cleavage on pro-cofactor/cofactor activity. We have previously shown that clotting activity is obtained upon coexpression of the amino terminal (92 kDa) heavy chain and carboxyl terminal (80 kDa) light chain proteolytic cleavage products as individual, secreted proteins without the909 amino acid central region (Burke et.al., J. Biol. Chem. 261, 12574, 1986). In the present work the thrombin cleavage sites in the heavy and light chains previously characterized by others (D. Eaton et.al., Biochemistry 25, 505, 1986) have been modified to remove these sites and the mutagenized gene reassembled into separate expression vectors for the two chains. Coexpression of wild type and mutant proteins in COS-7 cells has been characterized by coagulant activity, immunological assays specific for each of the two chains, and radioimmuno-precipitations. Alteration of the thrombin cleavage site in the heavy chain (Arg-372→ΔArg-372) leads to loss of coagulant activity, whereas another mutant Arg-372→Lys-372 shows 20-fold reduced activity. Radioimmunoprecipitations and RIA data show that this is not a reflection of reduced synthesis or increased degradation of the mutant polypeptides. These results suggest that Arg-372 is required for the efficient folding, assembly, or proteolytic activation of Factor VIII.
THE EFFECT OF LOW DOSE HEPARIN ON THE FEEDBACK ACTIVATION OF FACTOR VIII IN VIVO
Authors: Devilée P P , Fiolet H F B M , van Dam-Mieras M C E , HemKer H C
Published: August 2018

Heparin is a well established drug in the treatment and prophylaxis of thrombosis. However, the in vitro methods for thecontrol of heparin therapy (aPTT, anti-Xa, anti-II) often are not sufficiently sensitive to monitor low-dose heparin therapy. We used an assay that is based upon the in vivo activation of factor VIII at the site of skin punction: the factor VIII activity in blood samples taken from a capillary wound increases with time Hurlet-Birk Jenssen et al. Path. Biol. 24, 6 (1976).

Assay: 50 μl blood samples are taken 30, 60 and 90 sec after capillary puncture. The samples are diluted with 950μl, 10 mM sodium citrate, Mich, buffer and centrifugated(3 min, 4 °C). In the diluted samples the factor VIII activity is determined.

In healthy volunteers the increase in factor VIII activity in the successive samples is fairly constant (1.4 - 1.8 fold) in spite of distinct interindividual differences in the factor VIII level. In patients receiving continuous intravenous heparin (20000 IE/24 h) and in a hemophiliaA patient there was no increase in factor VIII activity with the time. In individuals receiving 5000 IE heparin subcutaneously the increase in factor VIII activity was dependent upon the time interval between heparin injection and sampling. There was almost no influence of heparin on the 30 s samples but the 90 and 150 sec. samples showed distinctly prolonged clotting times at sampling points from 2 to 4 hours after heparin administration. It was not possible to detect the heparin effectby an automated anti-IIa assay or in a prothrombin time assay because the heparinlevels were below the detection limit of the tests.

These results suggest that low-dose heparin therapy has a definite influence on the feedback activation of factor VIII even in concentrations that are not detectable with the usual heparin tests.
ACYL PLASMINOGEN STREPTOKINASE VERSUS HEPARIN IN RECENT ACUTE MYOCARDIAL INFARCTION. PRELIMINARY REPORT OF A FRENCH INTERUNIVERSITY TRIAL
Authors: Bassand J P , Machecourt J , Cassagnes J , Lusson J R , Bourlard P , Anguenot T , Faivre J , Wolff J E , Peycelon J
Published: August 2018

One hundred and twenty seven patients (pts) suffering from a first acute myocardial infarction (AMI) were included in a multicenter trial involving three University Hospitals and 22 community Hospitals without catheterization facilities. Patientswere randomly allocated within 5 hours following the onset of symptoms either to acyl plasminogen streptokinase (APSAC) 30 IU within 5 min followed 4 hours later by heparin at a dose of 500 IU/kg/day or to conventional Heparin (H) therapy 500 IU/kg/day. 63 pts received APSAC and 64 received H. Both APSAC and H groups were similar in age, location of AMI, Killip class and time of randomization. After initial therapy Pts were referred to the University Hospitals and submitted between Day 1 and Day 7 to a selective coronary angiography and immediate percutaneous transluminal angioplasty (PTCA) of the infarct-related artery if suitable for the procedure.4 APSAC pts and 2 H pts died during the hospital course (NS).The arterial patency rate determined on average 3.4 ± 1.2 days after the onset of symptoms was 88% in APSAC and 42% in H (P<.001). 15 APSAC pts and7 H pts were submitted to PTC A with one failure in each group. LVEF and akinetic score (AK) were determined from left ventricular angioeraDhy :

On Day 14, pts were submitted to Ti 201 scan and to radionuclide angiography (RNA). Results are not yet available.

Our results showed that early infusion of APSAC in AMI produced a high short term patency-rate of the IRA with a tendancy toward improvement of the left ventricular systolic function which failed to reach statistical significance because of the relatively small size of the cohort.
EFFECTIVENESS OF INTRAVENOUS THROMBOLYTIC TREATMENT IN ACUTE MYOCARDIAL INFARCTION:SHORT AND MEDIUM TERM PROGNOSIS
Authors: Mauri F
Published: August 2018

An unblinded trial of intravenous stretokinase in early acute myocardial infarction was planned to study wheter the drug produces a clinically relevant benefit in terms of reduction of in-hospital and one year mortality.

11806 pts in one hundred and seventy six coronary care units were enrolled over 17 months.Patients admitted within 12 h after o.nSet of symptons and with no contraindications to SK were randomized to receive SK in addition to usual treatment and complete data were obtained in 11712 for what concerns in hospital prognosis.At 21 days overall hospital mortality was 10.7% in SK recipients versus 13%,in controls,an 18% reduction(p=0.0002,relative risk 0.81).The extent of beneficial effect appears to be a function of time from onset of pain to SK infusion(relative risk 0.74,0.80, 0.87 and 1.19 for the 0-3,3-6,6-9 and 9-12 h subgroups).The data of 1-year follow-up concerning 11605 pts(95.3% of the whole population)were available up to December the 31st.1987.4333 pts out of the SK-treated group(74.0%)and 4219 out of the control one (72.1%)were alive,with a significant difference.These results document that the benefit produced by SK in the hospital period remains substantially unchanged.The differences in mortality in favour of SK vs.C remain highly significant specifically for the 0-3 and 3-6 hrs subgroups and is dramatic for patients treated between one hour from onset of symptoms. 503 out of the 637 treated with SK were alive at 1-year follow-up versus 443 out of 641 control group pts:the amplitude of the benefit seems to be further increased in this particular subgroup.The GISSI results document conclusively that an acute thrombolytic treatment with SK in AMI is effective in reducing mortality not only over the short,but also over the medium period.
THE DISTRIBUTION OF GP lb AND THE STABILITY OF THE PLASMA MEMBRANE ARE DEPENDENT UPON AN INTACT MEMBRANE SKELETON
Authors: Boyles J K , Fox JE B , Berndt M C
Published: August 2018

Platelets are know to have a cytoskeleton of actin filaments. We have presented evidence that they also have a membrane skeleton linked to the cytoskeletal filaments and that the membrane skeleton is linked to GP Ib-IX on the plasma membrane via actin-binding protein. In the current study, electron microscopy of thick (0.2 ym) epoxy sections was used to identify the distribution of GP lb. After various treatments, platelets were fixed and incubated with affinity-purified GP lb antibody and colloidal gold-labeled Protein-A. The entire cell surface was covered with a network of short intersecting chains of relatively evenly spaced gold particles. This was true of platelets in blood dripped directly from a vein into fixative, of washed discoid platelets, and of platelets activated by thrombin, ionophore, or cold under conditions in which aggregation did not occur. This pattern was not affected by the size of the gold label, the immunocytochemical protocol, or the fixative. The number of gold particles per cell was between 10,000 and 20,000, indicating a 1:1 ratio of label to GP lb. The distribution of GP lb was not affected by a level of cyto-chalasin B sufficient to disrupt the actin filaments of the platelet cytoskeleton. Proteolysis of actin-binding protein is known to be induced by treatment of platelets with dibucaine and by platelet activation (with either ionophore or thrombin) under conditions in which cell aggregation occurs. These same treatments caused GP lb to cluster. They also produced platelets with unstable membranes that vesiculated when the cells were subjected to shear force during centrifugation or osmotic-ally stressed during fixation. These studies show that both the distribution of GP lb and membrane stability are dependent upon the integrity of actin-binding protein and the membrane skeleton. In the high-shear environment of the blood vessel, the membrane skeleton and its linkage to GP Ib-IX and the cytoskeleton may be essential for proper platelet function.
IDENTIFICATION OF GLYCOPROTEIN lb IN “IN VITRO” TRANSLATES FROM ISOLATED HUMAN PLATELET mRNA
Authors: Wicki A N , Walz A , Clemetson K J
Published: August 2018

Platelet membrane glycoproteins play a crucial role in platelet adhesion and activation. To understand how they function it is of great importance to know their amino acid sequences and structures. It is rather difficult to purify membrane glycoproteins in amounts that are sufficient to determine their amino acid sequences by protein sequencing techniques. The easier way seems to be molecular cloning of the genes for these proteins.

Metabolically stable mRNA derived from nucleated megakaryocytes is known to be present in the anuclear human platelets. We have developed a purification method for the isolation of platelet mRNA. Starting with 100 units of blood platelets we isolated 0.5 mg of mRNA by guanidine chloride/lithium chloride/phenol extraction. Crude mRNA as well as oligo-dT purified, polyadenylated mRNA, was assayed for protein synthesis in a reticulocyte lysate translation system in the presence of different labelled amino acids. SDS-PAGE of nonreduced and reduced samples of the translation products showed molecular masses up to at least 200 kDa. Immunoprecipitation and affinity column chromatography with poly- and monoclonal anti platelet glycoprotein lb antibodies showed that a protein with a molecular mass of 60 kDa crossreacting immunologically with glycocalicin is specifically recognized by these antibodies. The 60 kDa protein seems to be the nonglycosylated form of GPIbケ.
CHEMICAL CROSS-LINKING OF HUMAN THROMBIN TO PLATELET MEMBRANE GLYCOPROTEIN Ib
Authors: Jandrot-Perrus M , Didry D , Guillin M C , Nurden A T
Published: August 2018

The interaction of thrombin with proteins at the platelet surface was assessed by chemical cross-linking with the membrane impermeable reagents DTSSP and BS3. Washed platelets were incubated with 125I-α-thrombin, 125I-TLCK-inactivated α-thrombin or 125I-γ-thrombin, and then with 0.1 mM DTSSP or BS3. The pattern of 3H-labeled glycoproteins (GP) was only slightly modified, with no measurable loss of GPIb, lib, Ilia, V and IX. Thrombin was detected in high molecular weight complexes migrating at the top of a 3 % acrylamide stacking gel and at the tap of the separating gel (7 %). In addition, a complex of Mr : 240 000 has been characterized. This complex was formed with 0.5 to 50 nM thrombin, after a short incubation time (30 sec) with active thrombin as well as with TLCK-thrombin. Hirudin added in excess before the crosslinker inhibited its formation. After analysis of soluble extracts by crossed Immunoelectrophoresis against a rabbit anti-whole platelet serum, 3 precipitates were labelled, among them the GPIIb-Illa complex and the most intense, a precipitate in the position of GPIb. Immunoprecipitation of GPIb using a murine monoclonal antibody allowed the isolation of the 240 000 complex. This complex was not observed with platelets from a patient with the Bernard-Soulier syndrome or with chymatrypsin-treated platelets lacking the outer portion of the GPIb αchain. 1125I-γ-thrombin was unable to bind to GPIb and form sucha complex. In each of these three examples of a defective binding, of thrombin toGPIb, the pattern of platelet aggregation and release reaction are characterized b a prolonged lag phase. Our results confirm the ability of GPIb to bindα-thrombin and support a role for this interaction in governing, the velocity of the platelet response.
OPPOSITIONAL REGULATION OF PLATELET CALCIUM FLUX BY cAMP-MEDIATED PHOSPHORYLATION OF GLYCOPROTEIN lb
Authors: Carroll R C
Published: August 2018

Glycoprotein (GP) lb, a surface receptor for von Willebrand's factor and thrombin, is composed of two disulfide-linked subunits (alpha - 130 kDa; and beta - 25 kDa). No significance has yet been attributed to the finding that the GPIb beta subunit is the only platelet glycopeptide which is phosphorylated in situ. GPIb can be quantitatively isolated from Triton X-100 wfibTeTlatelet lysates by monoclonal antibody (AN51) to GPIb and Protein A-Sepharose. Using this technique, I find that phosphorylation of the GPIb-beta subunit increased up to three-fold upon exposure of 32P-labeled platelets to agents which increase .platelet cAMP levels. In fact, this glycopeptide is identical to the phospho-peptide identified as 24P in a previous study (Cox et al., J. Cell Biol. 98:8-15, 1984). The phosphorylation of 24P was shown to correlate with cAMP-mediated reversal of platelet activation. This phosphopeptide has also been linked to enhanced calcium uptake by microsomes from platelets pretreated with cAMP elevating agents relative to untreated controls ( Fox et al_., Biochem. J. 184:651-666, 1979). I have further fractionatecTthese membrane fractions by centrifugation on 15% to 55% sucrose gradients and correlated a better than 5-fold enhanced calcium uptake with membrane fractions enriched in phosphorylated GPIb-beta. Confirming evidence was obtained using Bernard-Soulier platelets missing the GPIb complex. These platelets not only lack the 24P phosphopeptide, their activation is not readily reversible by cAMP elevating agents. These studies suggest a major role for GPIb in the inhibition/reversal of platelet activation by cAMP. My hypothesis is that GPIb phosphorylation either stimulates calcium efflux or blocks influx across the platelet plasma membrane.
S PROTEIN/VITRONECTIN NEUTRALIZES THE ANTICOAGULANT ACTIVITY OF GLYCOSAMINOGLYCANS IN THE INHIBITION OF THROMBIN BY HEPARIN COFACTOR II
Authors: Preissner K T , Sie P
Published: August 2018

The complement inhibitor S protein, which is identical to the adhesive protein vitronectin, functions as heparin-neutralizing factor by protecting thrombin against fast inactivation by antithrombin III. The interference of S protein with glycos-aminoglycan-catalyzed inhibition of thrombin by heparin cofactor II was investigated in a purified system. In the presence of 0.3 μg/ml heparin, or 0.5 μg/ml pentosan polyphosphate (SP 54), or 2 μg/ml dermatan sulfate, S protein induced a concentration-dependent reduction of the inhibition rate of thrombin by heparin cofactor II. This resulted in a decrease of the apparent pseudo-first order rate constants by about 17-fold (heparin), or about 7-fold (SP 54), but only by about 2-fold for dermatan sulfate at a physiological ratio of S protein to heparin cofactor II. Likewise, S protein significantly counteracted the anticoagulant activity of heparin and SP 54 bot not of dermatan sulfate when tested in an inhibition assay using various concentrations of glycosaminoglycans. For heparin, the activity of S protein at the point of 50% inhibition of thrombin was expressed in the range 0.06-0.6 μg/ml (0.01-0.1 U/ml) and for SP 54 in the range 0.3-2 pg/ml. Exposure of the glycos-aminoglycan-binding region of S protein by reduction and carb-oxymethylation of the protein even increased the neutralizing activity of S protein towards heparin and SP 54. S protein not only was found together with thrombin in a binary complex. S protein also became incorporated into a ternary complex with thrombin and heparin cofactor II as judged by crossed immunoelectrophoresis, regardless whether complex formation was initiated by heparin or dermatan sulfate. These findings underline the role of S protein as potent glycosaminoglycan-neutral-izing protein in plasma and as scavenger protein which may bind to enzyme-inhibitor complexes of the coagulation system.
INTERACTION OF S PROTEIN/VITRONECTIN WITH CULTURED ENDOTHELIAL CELLS: PROMOTION OF ATTACHMENT AND SPECIFIC BINDING
Authors: Preissner K T , Anders E , Müller-Berghaus G
Published: August 2018

The interaction of the complement inhibitor S protein, which is identical to the serum spreading factor, vitronectin, with cultured human endothelial cells of macro- and microvas- cular origin was investigated. Purified S protein, coated for 2 h on polystyrene petri dishes, induced concentration- and time-dependent attachment and spreading of human umbilical vein endothelial cells (HUVEC) as well as human omental tissqe microvasular endothelial cells (HOTMEC) at 37°C. With 3 × 105 cells/ml (final concentration) more than 50% of the cells attached within 2 h incubation at 0.3 - 3 μg/ml S protein. The effect of S protein was specific, since only monospecific antibodies against S protein prevented attachment of cells, while antibodies against fibronectin, fibrinogen or von Wille-brand factor were uneffective. The pentapeptide Gly-Arg-Gly-Asp-Ser, which contains the cell-attachment site of these adhesive proteins including S protein, inhibited the activity of S protein to promote attachment of endothelial cells in a concentration-dependent fashion; at 200 μM peptide, less than 10% of the cells became attached. Direct binding of S protein to HUVEC and HOTMEC was studied with cells in suspension at a concentration of 1 × 106 cells/ml in the presence of 1% (w/v) human serum albumin and 1 mM CaCl2 and was maximal after 120 min. Both cell types bound S protein in a concentration-dependent fashion with an estimated dissociation constant KD=0.2pM. More than 80% of bound radiolabelled S protein was displaced by unlabelled S protein, whereas binding was reduced to about 50% by the addition in excess of either fibronectin, fibrinogen, von Willebrand factor or the pentapeptide. These findings provide evidence for the specific association of S protein with endothelial cells, ultimately leading to attachment and spreading of cells. Although the promotion of attachment was highly specific for S protein, other adhesive proteins than S protein, also known to associate with endothelial cells, may in part compete with direct S protein binding.
THE CONTRIBUTION OF FIBRINOGEN Aα, γn, AND γ’ CHAINS IN SUPPORTING ADP-INDUCED PLATELET AGGREGATION
Authors: Amrani D L , Mosesson M W
Published: August 2018

Human plasma fibrinogen (ϕ) is heterogeneous with respect to its γ’ chain population (γn 93%; γ’ 7%) which differ in that C-terminal residues 407-411 of γn are replaced in γ’ by a unique 20 amino acid sequence. DEAE-cellulose chromatography yields two major $ peaks (peak 1 and peak 2 ϕ). Peak 1 ϕ molecules each contain two γn chains whereas peak 2 ϕ molecules are heterodimeric with respect to γn and γ’ chains. Using washed human platelets (PL), we confirmed that the number of peak 2 ϕ molecules binding to PL in the presence of ADP is about half the number of peak 1 ϕ molecules (18,962 ± 2,298 vs. 44,366 ± 16,096 molecules per PL) although Kds were similar − 0.20 µM and 0.26 µM, respectively. We also confirmed that isolated S-carboxymethyl (SCM)γn chains supported PL aggregation well (85% of peak 1 ϕ @ 0.94 mg/ml). In contrast to γn chains, SCM γ’ chains supported aggregation poorly (12% of peak 1 ϕ), and a 50:50 mixture of γn and γ’ supported aggregation to only 65%. Nevertheless, we found that peak 2 ϕ (n=15) supported aggregation well in the presence of ADP (5-20 mM) to an extent that was ≥ 93% of that of peak 1 ϕ (n=8). Since the C-terminal region of the Aγ chain has been shown to play a role in ϕ-PL interactions, we evaluated whether this region in peak 2 ϕ molecules was particularly important for maintaining its ability to support PL aggregation. Peak 2 ϕ from which C-terminal Aγ chain fragments had been removed by digestion with plasmin from >96% to 93 or 90% coagulability, showed decreased PL aggregation potential (71% and 54%, respectively) compared to comparably digested peak 1 ϕ (90% coagulable; 90% aggregation). In contrast to plasmin digested peak 2 ϕ, an unchromatographed plasma ϕ fraction (I-9) lacking C-terminal regions of Aγ chains but rich in γn chains, supported PL aggregation to the same extent as did intact peak 2 ϕ. These findings indicate that ϕ molecules containing Yγn chains can approach the aggregation potential of ϕ molecules containing 100% γn only if intact Aγ chains are also present.
EARLY (< 5 SEC) PHOSPHORYLATIONS OF PLATELET PROTEINS FOLLOWING ACTIVATION BY ADP AND ADRENALIN, SEPARATELY AND IN COMBINATION
Authors: Gear LR A , Freas D , Carty J D
Published: August 2018

Understanding the earliest events (< 1 sec) in signal transduction of platelets is important, since there is evicenee that “shape change,” aggregation and secretion can all begin within this period. We have employed a guenched-flow approach to study these early events and found that thrombin can induce rapid phosphorylation of myosin light-chain kinase (20K) and a 47K protein (Blood, 67, 1738, 1986). To investigate the role of rapid phosphorylations in platelet activation, we have studied the influence of adrenalin and ADP during early (0.3 to 5 sec) stimulation. Aggregation in washed human platelets was assessed by following the loss of single particles and phosphorylation by analysing 32P-labeled proteins after gel electrophoresis. 15 µM adrenalin (without ADP) did not initiate significant aggregation or phosphorylation of myosin light chain (MLC). Phosphorylation of the 47K protein was increased by 20% at 5 sec. 0.5 µM ADP did not induce significant aggregation, but increased phosphorylation of MLC by 130% and the 47 protein by 20%. The combination of 0.5 µM ADP and 15 uM adrenalin induced significant aggregation by 0,3 sec (7.6%), which increased to 25.6% by 5 sec. Interestingly, MLC or 47K protein phosphorylation was not increased above control levels. However, the phosphorylation of four other proteins (77K, 102K, 140K and 185K), which previously had been very rapid (<1 sec) and reversible with 0.5 µM ADP alone, was now maintained, peaking at 3 sec. 10 µM ADP caused small sustained increases in phosphorylation of the same proteins. Adrenalin also caused rapid increases in the phosphorylation of 27K, 213 and 250K proteins. High levels of ADP (10 µM) only increased the 213 and 250K proteins; therefore the 27K protein appears adrenalin specific. Analysis of these early platelet phosphorylations will help understand how they are linked to initiation and maintenance of aggregation. Supported by NIH HL-27014.
PROTEOGLYCANS AND SULFATED PROTEINS OF PLATELETS: CHARACTERIZATION AND RESPONSE TO THROMBIN AND ADP
Authors: Schick B P , Walsh C J , Jenkins-West T
Published: August 2018

The proteoglycans (PG) and sulfated proteins of guinea pig platelets were labeled in vivo by intraperitoneal injection of (35S)sulfate. At 3 days after injection, platelets contained 3 distinct populations of chondroitin-6-sulfate proteoglycans which together constitute about 65% of the cellular (35S) label. Most PG elute from a DEAE-Sephacel column with 4M Gdn HC1 (PG-1, 87%), and elute at Kav 0.12 on Sepharose CL-6B. The PG-1 can be resolved by SDS-PAGE into two fractions. The remainder (PG-2, 13%) elutes from the DEAE-Sephacel column with 4M Gdn HCl/2% Triton X-100 or 2% CHAPS, and has a Kav of 0.07 on Sepharose CL-6B. About 20-25% of the cell (35S) label elutes from DEAE-Sephacel in the wash-through or with 0.23M NaCl, and can be resolved by SDS-PAGE into at least 8 distinct bands which we have tentatively characterized as sulfated glycoproteins. The remainder of the (35S) is in low molecular weight (LMW) material which does not adhere to DEAE-Sephacel and has not been further characterized.

Platelets were treated with either thrombin or ADP, and the cells were then separated from the supernatant by centrifugation. The radiolabeled molecules in the supernatant and the cells were analyzed by DEAE-Sephacel and Sepharose CL-6B column chromatography. About 65% of the total cell (35S) was released from the cells by thrombin. Most of this radiolabel adhered to the DEAE-Sephacel column, and was found to be PG-1. The remainder of the released (35S) was about half the LMW material. In contrast, only 10-15% of the (35S)labeled material retained by the cells adhered to DEAE-Sephacel and was found to be PG-2. The remainder of the (35S)-labeled material retained by the platelets was the sulfated proteins and the LMW material. ADP caused release of about 15% of the (35S), and this was found to be in part PG-1 and in part the LMW material, but not PG-2. None of the (35S)-labeled molecules appeared to be degraded during platelet activation. We suggest that the PG-1 represent the a-granule and PG-2 the membrane proteoglycans. The sulfated proteins have not been described previously. Their role is not known, but we hypothesize that they may form part of the negative charge of the glycocalix and thus be part of the reactive surface of the platelet.
APPLICATIONS OF PLASMA ASSAYS OF CROSSLINKED FIBRIN DEGRADATION PRODUCTS (XLFDP) IN THE DIAGNOSIS OF THROMOEMBOLIC DISEASE
Authors: Whitaker A N , Masci P , Rowbotham B , Reasbeck P , Guerrini S M , McCaffrey J , Lee K , Elms M J , Bunce I H , Webber A J , Wylie F , Rylatt D B , Bundesen P G
Published: August 2018

The presence of XLFDP in plasma, a consequence of the balance between fibrin generation and dissolution, is an indirect index of the presence of fibrin and raised levels accompany intravascular thrombosis. The potential of an enzyme immunoassay (EIA) for XLFDP, using the monoclonal antibody DD-3B6/22 was evaluated in 104 patients with suspected DVT receiving lower limb venography. Using a reference level of 400 ng/ml of XLFDP, all patients with a positive venogram had elevated levels (sensitivity 100%), although levels were also elevated due to causes other than DVT in some patients with negative venograms (specificity 52%). Higher elevations of XLFDP occurred in proximal than in calf DVT.

A second study of another 48 patients receiving venography for suspected DVT confirmed the sensitivity of EIA while the complementary latex "Dimertest", also based upon DD-3B6/22, was positive in 19 and negative in 3 patients with confirmed DVT. In an ongoing study, 130 patients have been followed after abdominal operations with 125I-fibrinogren leg scanning and venography when appropriate. Levels of XLDFP (EIA) were elevated in all patients developing thrombosis. Latex assays were positive in 42/48 patients with positive leg scans. While elevated levels of XLDFP do not specifically define the location or even the presence of a thrombus they reliably indicate the possibility of thrombosis in patients without antecedent medical or surgical illnesses. In complicated surgical patients,an arbitrary cut-off of 1000 ng/ml is more appropriate for screening for the possibility of DVT. A positive latex test is also suggestive, although it is negative in some patients with calf thrombi. Measurements of XLFDP, with high sensitivity but moderate specificity, are potentially valuable in selecting patients for invasive investigations such as venography, and combinations with other noninvasive screening tests for require evaluation
PLASMA LEVELS OF FRAGMENT D-DIMER OF CROSSLINKED FIBRIN DURING THROMBOLYTIC THERAPY WITH RECOMBINANT TISSUE-TYPE PLASMINOGEN ACTIVATOR
Authors: Declerck P , Mombaerts P , Holvoet P , Collen D
Published: August 2018

Plasma levels of crosslinked fibrin degradation products (XLDP) were measured before and at the end of the administration of rt-PA (40 to 100 mg over 1.5 to 8 hours) in healthy volunteers (n=5) and patients with deep venous thrombosis (DVT) (n=8), pulmonary embolism (PE) (n=16)and myocardial infarction(MI)(n=10). Determinations were performed using our newly developed ELISA, specific for crosslinked fibrin derivatives, based on two monoclonal antibodies (15C5 and 8D3H2) raised against purified human fragment D-dimer. All plasma samples were collected on citrate and trasylol. Results are expressed as mean and range of D-dimer equivalents (μg/ml).

Baseline levels in patients with MI are only slightly elevated. The increased levels inDVT and PE are in agreement with previous studies. After infusion of rt-PA a small increase of XLDP is seen even innormal subjects. A very marked increasof XLDP is detected in patients with PE and DVT but not in patients with MI. This may reflect differences in the amounts of fibrin clot dissolved in these patient groups.

No significant correlation was found between the increase of XLDP and success of therapy, although a significant difference in D-dimer levels was formed between the two groups with PE: successful (n=ll): 116 (range 61-192) vs. unsuccessful (n=5): 68 (36-155).

Thus, XLDP are already elevated under baseline conditions in patients with DVT and PE and increase very markedly during thrombolytic therapy. The absolute levels after thrombolytic therapy do not strictly correlate with success of therapy. It could be useful to measure D-dimer levels during early stages of therapy, because the rate of increase of XLDP levels might correlate with the efficacy of thrombolytic treatment.
FIBRIN DEGRADATION PRODUCTS (FbDP) IN HEALTHY VOLUNTEERS AFTER INFUSION OF RECOMBINANT TISSUE-TYPE PLASMINOGEN ACTIVATOR(rt-PA)
Authors: Seifried E , Rijken D C , Hoeqee B , TansiAiell P , Kluft C , Nieuwenhuizen W
Published: August 2018

During thrombolytic therapy of myocardial infarction (MI) with urokinase or streptokinase (SK), levels of fibrin(ogen) degradation products in serum are often dramatically elevated as a result of a combination of systemic fibrinogenolysis and local thrombolysis. Others have measured increased levels of D-dimer in serum of MI patients after SK therapy and postulated that thrombolysis could be monitored during SK therapy by measuring D-dimer levels. In the present study rt-PA was infused into healthy volunteers to analyse if elevated FbDP levels in MI patients really reflect coronary thrombolysis or could be due to a systemic effect. Over a period of 60 min., three groups (n = 6 each) were given i.v. 0.23 mg rt-PA/'kg (group I), 0.50 mg rt-PA/kg (group II) and a placebo infusion (group III), respectively. Two blood samples were taken from an anticubital vein in the arm contralateral to the site of infusion (one on citrate/ aprotinin, the other on citrate alone) at different time points. Using a new enzyme immunoassay (EIA), based on monoclonals and development by us, we measured FbDP in plasma (not serum). Before infusion all volunteers had FbDP levels ≪ 0.5µg/ml. Upon infusion FbDP levels in groups I and II increased to average values of 1.0 ± 0.4/µg/ml and 0.8 ± 0.2/µg/ml, respectively, for the samples taken in citrate/aprotinin. The values in citrate alone did not differ significantly, and were 1.1 ± 0.5/µg/ml and 0.8 ± 0.3/µg/ml for groups I and II, respectively. FbDP levels in group III remained ≪ 0.5/µg/ml. The results show that FbDP levels increase upon rt-PA infusion, even in healthy volunteers. This suggests lysis of systemic fibrin. We conclude that lysis of systemic fibrin limits the value of FbDP levels as a measure for coronary thrombolysis.
DISTINCTION BETWEEN ANTI CARDIOLIPIN (aCL) ANTIBODIES IN SYPHLIS AND THE "ANTI PHOSPHOLIPID SYNDROME"
Authors: Harris EN , Wasley G , Hughes GRV
Published: August 2018

Patients both with syphilis and with the Anti-Phospholipid Syndrome (APS) produce antibodies that bind cardiolipin (CL). However, thrombosis, fetal loss, and thrombocytopenia, as well as lupus anticoagulant (LA) activity occur in APS but not syphilis patients. Differences between aCL antibodies produced in these 2 disorders have been suggested but not proven. We report a newly devised ELI9A assay using carbon coated VDRL (C-VDRL) as antigen, as well as a variety of other ELISA assays with various phospholipids as antigens, to study sera from 20 syphilis and 10 APS patients. Inhibition experiments were alSo performed to support findings with the ELISA assays.
Syphilis sera bound both CL and C-VDRL coated plates, but binding to C-VDRL was always greater. APS sera bound CL but not C-VDRL plates. When plates were coated with low concentrations of negatively charged PLs, syphilis sera bound CL almost exclusively but APS sera bound negatively charged phospholipids (PLs) as well as they did CL. Inhibition experiments confirmed the high avidity of syphilis sera for CL, particularly when presented as the VDRL antigen; but APS sera bound negatively charged PLs as avidly as CL. Neither syphilis nor APS sera bound zwitterionic phospholipids.
These findings may be important in that CL is limited primarily to mitochondrial membranes, but negatively charged PLs are widely distributed in cell membranes of the body. Hence, autoimmune sera which binds negatively charged PLs as well as CL may affect cell function more readily than syphilis sera which is limited primarily to CL binding. Too, negatively charged PLs (but not CL) are present in PL mixtures used in the LA test, hence APS (but not syphilis) plasma would more likely show LA activity. The genetic origins of these 2 groups of antibodies may also be different although they bind related antigens.
EFFECT OF INTERFERON GAMMA ON PROCOAGULANT ACTIVITY FROM HUMAN PROMYELOCYTIC CELL LINE (HL 60)
Authors: Gamba G , Dezza L , Montani N , Gangale G , Gangale G , Ascari E
Published: August 2018

Interferon gamma (IFN), a lymphokine acting as biological response modifier, can induce morphological and phenotypic differentiation of some leukemic cell lines, specially along the monocytic pathway.

Furthermore, myeloid leukemic cells and normal monocytes have been demonstrated to possess procoagulant activity (PCA).

The aim of this study was to investigate the modulation of PCA induced by treatment with IFN on human promyelocytic cells from HL 60 cell line.

Cells were cultured in suspension for 5days in RPMI 1640 medium supplemented with 10% fetal calf serum in the absence or in the presence of different concentrations of IFN (100-10.000 u/ml) at37°C and 5% CO2

Differentiation was assessed by morphological and cytochemical methods (MGG, ANAE, CAE, MPO) on cytospin preparations and surface marker analysis with monoclonal antibodies (0KMI, Mo2, MY9, MY7, MY4).

PCA was measured as capacity to shortening ricalcification time of normal plasma and of factor VIII, VII and X deficient plasmas by the cells and the conditioned media.

Untreated HL 60 cells exhibit high tissue factor-like PCA, related to the cellnumber. IFN treatment (1000 u/ml) induced at the same time, monocytic differentiation and significant increase in PCAboth in the cells and in the conditioned media.

The PCA was not further affected by higher concentrations of IFN, unabletodetermine cell maturation.

In conclusions the modulation by IFN seems to be dependent onmonocytic differentiation of HL 60 cells.
DEFECTIVE GENERATION OF PROCOAGULANT ACTIVITY BY TUMOR-ASSOCIATED MACROPHAGES EXPOSED TO DIFFERENT STIMULI
Authors: Semeraro N , Errori A , Casali B , Dontati M B , Mantovani A
Published: August 2018

Mononuclear phagocytes, an integral part of the lymphoreticular infiltrate of human and experimental tumors, might contribute to fibrin deposition within malignant tissues through the production of procoagulant activity (PCA). We have studied the PCA of tumor-associated macrophages (TAM) in two poorly immunogenic, metastatic murine sarcomas (mFS6 and MN/MCA1); peritoneal macrophages (PM)from tumor-bearing and control animals were also studied, as reference cell populations. TAM were prepared from disaggregated tumor tissue and PM from ‘lavage’ fluid by adherence to plastic.PCA was evaluated by a one-stage clotting assay immediately after isolation (basal PCA) and following in vitro stimulatio.Basal PCA was very low(<1 u/104 M) inall cellpreparations. Exposure of PM from both normal and tumor-bearing animals to endotoxin (S. enteritidis LPS, 1 ug/ml f..), phorbol myristate acetate (PMA,0. ug/ml) or the chemotactic peptide FMLP(10-7 M) resulted in a 10-,7- and 3-fold increase of PCA respectively. In contrast TAM from mFS6 and MN/MCA1 consistently failed to generate PCAin response to different concentrationsof the same stimuli. Treatment of TAM with aspirin (10-3M) did not affect the cell unresponsiveness. Fluorescence microscopy showed that almost all PM were stain ed with fluorescein isothiocyanate (FITC)-LPS, while 10% of the TAM were stained. Moreover binding studies demonstrated that TAM had a loyer number of specific binding sites for phorbol esters than PM. These data suggest that the defective responsiveness of TAM to endotoxin, PMA and, possibly, to FMLP is due to the lack or very low expression of binding sites for these agents on the cell surface. The tumor environment may orient the functional status of in situ macrophages in a sense less favourable to the host.
ANTITHROMBIN III CONCENTRATE (AT-III) FOR PROPHYLAXIS ANDTREATMENT OF CONGENITAL AND ACQUIREDAT-III DEFCIENCY
Authors: Schwartz R , Kavanagh E , Bauer K , Rosenberg R , Ballard J , Latino J , Strother V , Mosesson M , Haire W , DeLeo M
Published: August 2018

An investigation has been undertakenin 13 patient studies to determine the efficacy of AT-III in prevention and treatment of thrombosis in patients with congenital (cong.) and acquired (acq.) AT-III deficiency. The mean in vivo incremental recovery of 17 infusions of AT-III in 7 patientswith cong. AT-III deficiency (2 kinetic studies, 5 prophylaxis) was 1.4%/U/kg (functional assay) administered. The mean in vivo recovery of 38 infusions in 4 non-bleeding patients(3 cong., 1 acq.) treated for thrombosis or pulmonary embolism (P.E.) with heparin was 1.33%/U/kg which was not significantly different. The half-life determined in an earlier study exceeded 2.5 days. All patients treated prophylactically or therapeutically received a loading dose to increase plasma AT-III to 120%, and then received maintenance doses, generally every 24 hours, to maintain plasma AT-III levels in the general range 80-120%. AT-III levels were monitored every 12 hours initially and doses and intervals modified accordingly. None of 5 patients with cong. deficiency treated prophylactically for high risk situations (surgery, delivery, catheterization) developed a thrombotic complication. Six patients (3 cong., 2 acq., 1 probably acq.) were treated for thrombosis/P.E., two of whom were pregnant. Heparin resistance was reversed in two, both pregnant. One patient with superior mesenteric-portal vein thrombosis (cong. deficiency) and another with superior mesenteric artery-aortic occlusion (acq. deficiency) survived without further progression of thrombosis. DIC resolved in one patient treated with AT-III and benefit was observed in a 2nd patient as well. AT-III was well tolerated, with but a single mild reaction in 176 infusions. We conclude AT-III may be beneficialas prophylaxis in patients with cong.AT-III deficiency during high thrombotic risk situations, as well as anadjunct to heparin in treatment for thrombotic complications in congenital and acquired AT-III deficiency.
ANTITHROMBIN ROUEN-I(47 ARG→HIS) AND ROUEN-II (47SER) : TWO NEW VARIANTS WITH DECREASED HEPARIN AFFINITY
Authors: Borg J Y , Owen M C , Soria C , Caen J , Carrell R W
Published: August 2018

Four families with AT-III variants of decreased heparin affinity have been compared with families with a quantitative deficiency of AT-III. The affinity variants had a lower incidence of thrombosis and, unlike thedefiency variants, had no increase in plasma FDF (defined by anti-D-neo ELISA assay). These results support a major physiological role for the progressive activity of AT-III.

The characterisation of the affinity variants provides information onthe heparin-binding site. Variants were isolated from plasma of heterozygotes by NaCl elution gradient from a heparin-Sepharose column. Maps of tryptic and SA V8 peptides were compared on TLC and HPLC with analysis of aberrant peptides. Confirmation was by gas-phase automated sequencing.Two variants were unequivocally identified.

1) Rouen-I (47Arg→His) wasfoundin a heterozygote with a history of a single cerebral occlusion.

2) Rouen-II (47Arg→Ser) was in a forty years old heterozygote with an acute myocardial infarction but no other history of thrombosis.

Three mutations of Arg47 are known: Cys(Toyama), His(Rouen-I) and Se(Rouen-II) all with decreased heparinaffinity.

Functional and structural modelling studies are reported indicating apositive heparin-binding site formedby the A and D helices centred on Arg47 (A2). The mutation in Rouen-IIcreates an unutilised oligosaccharide attachment Asn⋆-Arg-Ser suggesting facultative glycosylation. This would, as an incidental observation, support a translational (pre-secretory) origin of the high affinity AT-III beta.
HEPARIN INDEPENDENT PURIFICATION OF ANTITHROMBIN III (AT III) BY IMMUNO-AFFINITY CHROMATOGRAPHY RESULTING IN A FUNCTIONALLY INTACT MOLECULE
Authors: Jørgensen M
Published: August 2018

Previous methods for purification of AT III are based on its heparin-binding capacity. However, in congenital AT III deficiency abnormal inhibitor molecules with impaired binding of heparin and/or thrombin has been reported. The aim of the present study was to develop a purification method based on immuno-affinity chromatography, and thus independent of the heparin binding capacity.

Rabbits were immunized with human AT III purified by a three-step procedure involving dextran sulphate precipitation, affinity chromatography on heparin-Sepharose and ion-exchange chromatography on DEAE-Sephadex A-50. Rabbit immunoglobulins against human AT III were isolated by affinity chromatography using purified human AT III coupled to CNBr-activated Sepharose 4B. Trace amounts of immunoglobulin against human albumin, IgG and IgM were removed by solid phase immunoadsorption. The highly purified immunoglobulins against human AT III were coupled to CNBr-activated Sepharose 4B. This anti-AT III-Sepharose was used for single-step purification of AT III from plasma. Elution was performed by Na-citrate buffer at pH 3.0 and the eluted fractions immediately neutralized. The recovery was more than 60%.

The purified AT III appeared as a single protein band in SDS-poly-acrylamide gel electrophoresis with or without reduction. Affinity purified AT III and AT III purified by the three-step procedure were indistinguishable when analyzed by crossed immunoelectrophoresis in the absence and the presence of heparin isoelectrical focusing in polyacrylamid gel at a pH 4-6.5 gradient, and SDS-polyacrylamide gel electrophoresis. AT III antigen concentration was determined by electroimmunoassay and the reactive site concentration determined by titration with purified human thrombin using Phe-Pip-Arg-Nan (S-2238) as substrate. The ratio (active site conc.)/(antigen conc.) was identical in the two AT III preparations. It is concluded that this single-step immuno-affinity chromatography gives a high recovery from plasma of a highly purified functionally intact AT III molecule. The purification method is independent of the heparin binding capacity of AT III. This is of particular importance for the purification and characterization of abnormal AT III molecules with impaired heparin-binding site.
LMW-HEPARIN VERSUS HEPARIN-DHE IN ORTHOPAEDIC SURGERY
Authors: Breyer H G , Rahmanzadeh R , Bacher P , Werner B
Published: August 2018

The efficiancy and the side effects of a LMW heparin (FRAGMINR, KabiVitrum) and Heparin-DHE (Sandoz) have been compared in a randomized open prospective study of 120 patients (60/60) undergoing elective surgery on the lower limbs (total hip and knee replacement, corrective osteotomies). A radiofibrinogen uptake test (RFUT) was regularly done on all patients. Positive tests were controlled by ascending phlebography. The parameters, clinically obtained, included the intra-and postoperative blood loss, wound closure, and the incidence of haematoma. Hb, Hk, red and white blood cells, thrombocytes, total protein, aPTT, AT III, TT, and anti-Xy-activity were analyzed at the day before operation, the 2nd, 4th, and 6th day after operation.

There were three positive RFUT in the group of LMW heparin (5 per cent), and there were six (10 per cent) in the control group. No pulmonary embolism occurred. In no case an operative treatment of deep vein thrombosis was done. There were no statistically significant differences in intra- and postoperative blood loss, and in the laboratory data, except the anti-Xa-activity, which was significantly higher in the LMW heparin group.

The comparative study has shown, that a single daily injection of LMW heparin (FRAGMINR) is more effective than the two daily injections of the combination of UF heparin and DHE in order to prevent postoperative thromboembolism
ENOXAPARINE (LOVENOXR), VERSUS STANDARD HEPARIN IN PROPHYLAXIS OF DEEP VEIN THROMBOSIS (DVT) AFTER TOTAL HIP REPLACEMENT (THR)
Authors: LE BALC’H T , LANDAIS a , BUTEL T , WEILL D , PASCARIELLO J C , PLANES A
Published: August 2018

THR is associated with a high risk of thromboembolic complications. Enoxaparine, LovenoxR, a low-molecular-weight-heparin, and standard heparin were compared in their abilities to prevent DVT in patients undergoing THR. The efficiency and the bleeding risk of each treatment were studied.

237 patients, with a non traumatic hip disease, requiringTHR, were included in a multicentric, randomized, double blind trial. Mean age:65.8 years ± 9.2; mean weight :67.3kg ± 1.3.

113 patients received standard heparin, 5000 UI/8 hrs, by"subcutaneous (SC) injection. 124 patients received Enoxaparine, 40 mg/24 hrs, by SC injection. Administration of drugs was begun 2 hours before operation for standard heparin, 12 hours before operation for Enoxaparine. Patientswere treated for 10-15 days, until bilateral ascending phlebography (BAP) had been completed.

Lower limbs BAP were performed in 228 patients. The incidence of DVT was significantly lower in the Enoxaparine group : a DVT was detected in 15(12.5 %) of 120 patients who received Enoxaparine and in 27(25%) of 108 patients who received standard heparin (p=0.014).A pulmonary embolism occurred in 1 patient of the heparin group, in none of the Enoxaparine group.

The frequency of bleeding complications was significantly lower in the Enoxaparine group. A post operative wound hematoma occurred in 1 patient of the Enoxaparine group and in 3 patients of the heparin group. Red cell transfusions requirements were significantly lower in the Enoxaparine group (3.37 U ± 1.81) than in the heparin group (3.84U ± 1.70)(p=0.03). The hemoglobin level was significantly higher, on the 3rd, 4th post operative day, in the Enoxaparine group.

Subcutaneous Enoxaparine (40 mg/24 hrs) was significantlymore efficient than subcutaneous heparin (5000 UI/8 hrs) in preventing DVT, in patients undergoing THR. The incidence of bleeding complications was significantly lower in the Enoxaparine group.

Enoxaparine (LOVENOXR) - PHARMUKA S.F.