bluuebunny/crossref_metadata_2025 huggingface dataset:
FIBRINOGEN RECEPTORS IN BLOOD PLATELETS OF MIGRAINE PATIENTS
Authors: Valkowiak B , Kozubski V , Pawlowska Z , Ciernlewski C S
Published: August 2018

The augmentation of platelet aggregability in migraine patients was found in many studies, by the use of both in vivo and in vitro techniques. In order to evaluate the platelet characteristics responsible for the increased aggregability of migraine platelets we determined their binding capacities and the apparent dissociation constant (KD
app) of fibrinogen receptors. Twelve non-pregnant women in age ranged from 20 to 44 years with unequivocal history of common migraine and 10 control healthy age matched women were used in these studies. The patients were assessed in headache-free intervals. Blood was drawn into acid citrate dextrose containing apyrase (0.1 mg/ml) and platelets were isolated by differential centrifugation. The mean number of platelet fibrinogen receptors exposed by ADP in migraine patients ( 65197 ± 6276 ) was statistically ( p<0.05 ) higher than that obtained in healthy controls ( 40217 ± 7678 ). The apparent Kd for fibrinogen receptors in migraine platelets (6.01 ± 1.11 × 10−7 M ) was lower than that in control women about threefold < 2.17 ± 0.56 × 10−6 M). The difference was statistically significant ( p<0.02 ). With these results we conclude that the increased capacity and binding affinity of fibrinogen receptors may be responsible for the elevated number of circulating platelet aggregates in migraine patients and for the prevalance of various kinds of strokes during migraine attack.
BINDING OF FIBRINOGEN TO PLATELET GLYCOPROTEIN (GP) IIb/IIIa IS CRUCIAL FOR SHEAR-INDUCED PLATELET AGGREGATION
Authors: Ikeda Y , Murata M , Araki Y , Yamamoto M , Watanabe K , Ichitani M , Sakai K , Itagaki I , Mori Y
Published: August 2018

It is well known that human platelets can aggregate in vitro under certain shear stress without adding aggregating inducers. However, the mechanism of this shear-induced platelet aggregation has not been clarified yet. In this paper, we have investigated the role of fibrinogen and GP IIb/IIIa in shear-induced platelet aggregation. Citrated human platelet-rich plasma (PRP) was subjected to controlled shear stress levels in a polycarbonate cone and plate viscometer at 37°C for 2 minutes. After shearing the particle count was measured by an electronic particle counter. Particles with sizes from 3 to 20 μ cum were considered as single platelets. In unsheared PRP most of the particles were single platelets, but platelet doublets and platelet fragments larger than 3 μ cum were also counted. After exposure to shear rate of 3,600 - 9,000 sec−1 , the particle counts were decreased in a shear rate dependent manner, while LDH leakage from platelets was not significantly increased and 3H-serotonin release was 2-7%. Scanning electronmicroscopy clearly showed the presence of large platelet aggregates when the particle counts were decreased. Platelets from two patients with thrombasthenia and one patient with afibrinogenemia, however, failed to aggregate at a shear rate of 9,000 sec−1. Shear-induced aggregation was inhibited by monoclonal antibody to GPIIb/IIIa (1 μg/ml) and synthetic peptide, Arg-Gly-Asp-Ser, (1 mM). When fibrinogen was added to PRP from a patient with afibrinogenemia, shear-induced aggregation became evident as seen in normal platelets. Apyrase and hirudin showed no effect on shear-induced aggregation. Indomethacin (100 μM) and TXA2 synthetase inhibitor, OKY-046 (100 μM) markedly inhibited aggregation, while TXA2 competitive inhibitor, ONO-3708 (100 μM) exhibited only partial inhibition.

Our results indicate that binding of fibrinogen to GPIIb/lIIa is also crucial for shear-induced platelet aggregation and that the exposure of fibrinogen receptor on GPIIb/IIIa may partially depend upon TXA2 synthesis in platelets.
PRODUCTS OF LIMITED PROTEOLYSIS OF GLYCOPROTEIN Ilia ON TEE PLATELET MEMBRANES
Authors: Niewiarowski S , Eckardt A , Lukasiewicz H , Norton K , Huanq Tur-Fu , Komecki E
Published: August 2018

Previous investigations from our laboratories have demonstrated the limited proteolysis by chymotrypsin of GPIIIa and the formation of a major component migrating on SDS polyacrylamide gel electrophoresis with an apparent molecular weight of 60 kDa in a nonreduced system and 66 kDa in a reduced system. The formation of this component occurred in parallel with the express ion of fibrinogen receptors on the platelet surface. We raised in rabbits a monospecific polyclonal antibody against highly purified 60 kDa ccmponment. Using available rabbit polyclonal antisera (anti-GPIIIa and anti-66 kDa) and murine monoclonal anti GPIIIa antisera (AP3 and SSA6) a caiplete imnunological crossreactivity between GPIIIa and 60 kDa was established. The limit of detection by immunoblotting assay was 20 ng of GPIIIa or 60 kDa in Triton X100 extract of platelets. Extracts of intact platelets, of AEP- or thranbin-stEmulated platelets separated on SDS PAGE showed a strong band corresponding to GPIIIa, a light band migrating with an apparent molecular weight of 120 kDa and no detectable 60 kDa ccnponent. Incubation of platelets with chymotrypsin, pancreatic elastase or human granulocyte elastase resulted in the appearance of 60 kDa and in a progressive increase of 120 kDa component detectable by all polyclonal and monoclonal antisera. In contrast to 120 kDa, reduced GPIIIa (108 kDa) was not recognized by AP3 and SSA6. The 120 kDa did not dissociate in the presence of 6M urea and 5 mM EDTA. The component migrating with an apparent molecular weight of 120 kDa also appeared during prolonged storage of purified 60 kDa ccnponent. The partial purification of 60 kDa and 120 kDa components together with GPIIIa was accomplished by Con A sepharose chromatography. In conclusion 60 kDa and 120 kDa platelet membrane components represent products of proteolysis of GPIIIa.
PLATELET ADHESION ON SYNTHETIC SURFACES PRETREATED WITH DILUTED PLASMA IS DETERMINED BY THE SURFACE CONCENTRATION OF "NATIVE" FIBRINOGEN
Authors: Lindon J N , Kushner L , Shiba E , Salzman E W
Published: August 2018

Platelet adhesion and activation on synthetic surfaces are thought to require the prior adsorption of fibrinogen. We have reported that platelet activation by polyalkyl methacrylates (measured in bead columns exposed to flowing blood) is better correlated with the concentration of conformationally unaltered, "native" (recognizable by radiolabeled antifibrinogen antibodies) bound fibrinogen than with total bound fibrinogen (measured with radiolabeled fibrinogen) following exposure of the surfaces to purified fibrinogen in solution or to diluted blood plasma (Blood 68:355, 1986). We now report that adhesion of washed platelets to polybutyl methacrylate (PBMA; approx. 20% retention in bead columns), was unaffected by preincubation of the surface with whole plasma but was increased significantly by precoating with diluted plasma, with maximum retention (approx. 65%) occurring with plasma diluted 3000-fold. Upon exposure of PBMA to various plasma dilutions the surface concentration of antibody-detectable fibrinogen, but. not of total surface-bound fibrinogen, was correlated with the activation of washed platelets by such pretreated surfaces, With maximal platelet reactivity occurring in columns precoated with the plasma dilution (1:3000) that produced the highest concentration of "native" surface-bound fibrinogen. The plasma dilution that gave maximum total fibrinogen adsorption (100-fold dilution) was not correlated with the concentration of antibody-detectable fibrinogen. Fab fragments of polyclonal anti-fibrinogen antibodies totally prevented platelet activation by PBMA surfaces precoated with diluted plasma. It appears that participation of surface-bound fibrinogen in platelet activation on some artificial surfaces requires that fibrinogen be adsorbed in a conformationally "native" state, presumably thereby permitting multivalent, cooperative interactions with GP IIb/IIIa receptors on platelets.
ADHESION OF BLOOD PLATELETS TO THE EXTRACELLULAR MATRIX OF CULTURED CELLS IN WHICH COLLAGEN SYNTHESIS IS IMPAIRED OR INDUCED
Authors: Hindriks G A , de Boer H C , Nieuwenhuis H K , Sixma J J , de Groot P H G
Published: August 2018

One of the first phenomena in the hemostatic plug formation is adhesion of platelets to the subendothelium or to the perivascular connective tissue. With a rectangular perfusion chamber (Sakariassen et al., J.Lab.Clin.Med.(1983) 102, 522-535) it is possible to study platelet adhesion in flowing blood to extracellular matrix (ECM) of cultured cells.

Collegen type I and III play an important role in the adhesion of platelets. To study their role in more detail, cultured cells with changed collagen synthesis were used. For this purpose, we used fibroblasts of a patient with Osteogenesis Imperfecta type I (0.I.)(characterized by impaired collagen synthesis) cultured from a skin biopsy and used in the fourth passage. Platelet adhesion to ECM of 0.1. fibroblasts was decreased in comparison to ECM of normal fibroblasts (8% versus 17% at low shear rates (300 s™1 ) and 5% versus 24% at high shear rates (1300 s™1 ) after perfusion with whole blood for 5 min). No aggregate formation occurred on patient’s matrices, whereas small aggregates were observed on ECM of normal fibroblasts. Addition of ascorbic acid to culture medium of fibroblasts, smooth muscle cells and endothelial cells causes increased collagen synthesis with increased fibril formation. Platelet adhesion to ECM of endothelial cells showed an increased adhesion of 25% only at high wall shear rates (1300 s™1 ). Induction of collagen synthesis in smooth muscle cells resulted in a strong increase of thrombus formation on their matrices. These data indicate that platelet adhesion to the matrix of cultured cells is strongly dependent on the quantity and nature of the reactive collagens.
MAPPING OF 6 MONOCLONAL ANTIBODIES TO HUMAN FACTOR IX
Authors: Frazier D , Wha Lin Shu , Ware J , Smith Kenneth , Reisner Howard , DeSerres M , Wallmark A , Ljung R , Nilsson I M , Stafford D W
Published: August 2018

In order to map the regions of human factor IX recognized by monoclonal antibodies we have inserted random fragments of the coding region of the cDNA for human factor IX into the lambda phage expression vector, lambda gtll. The resultant recombinant phage were screened with monoclonal antibodies, the immunoreactive phage were isolated, and the DNA of the inserted fragment was sequenced to determine which amino acids were immunoreactive. This data, coupled with data obtained from the use of specific fragments of human factor IX expressed in E. coli from a T7 phage promoter, has allowed us to map the location of several epitopes on the surface of the factor IX molecule. In those cases where the antibodies are specific for human factor IX, additional narrowing of the epitope is possible by comparing the amino acid sequence of human factor IX to the bovine molecule. Six monoclonal antibodies from 3 different laboratories have been mapped. The immunodominant epitopes appear to be the amino terminus of the activation peptide, the amino terminus of the heavy chain and the epidermal growth factor domains.
EFFECTS OF CONSTANT INFUSION OF ILOPROST, A STABLE PROSTACYCLIN DERIVATIVE DURING CARDIOPULMONARY BYPASS
Authors: Walker Isobel D , Davidson J F , Wheatley D J , MacArthur K , Spyt T J
Published: August 2018

Prostacyclin (PGI2) is a potent inhibitor of platelet aggregation but it is highly unstable. Iloprost (Schering A.G.) is a stable carbacyclin derivative of PGI2 which is also a potent inhibitor of platelet aggregation. The effect of constant Iloprost infusion during cardiopulmonary bypass (CPB) on blood loss, platelet numbers, secretory proteins and platelet sequestration was studied in 50 adult males undergoing CPB for elective coronary vein bypass graft surgery.

In a double-blind, randomised, placebo-controlled study (25 patients in each group) intravenous infusion of Iloprost was commenced at 5ng/kg/min immediately after the induction of anaesthesia and increased to 10ng/kg/min when the patient was established on CPB. The infusion was discontinued on completion of CPB. Blood loss and units of blood transfused were not significantly different in the two treatment groups. During CPB, mean platelet numbers fell significantly in both groups but were significantly higher (P<.001) in the Iloprost group than in the placebo group at the end of CPB and until 18-24 hours post CPB (P<.05). Mean plasma thromboxane B2 and β thromboglobulin levels were marginally lower (P<.05 and P<.02) in the Iloprost group but the distribution of platelet factor 4 results were similar in both groups. Post operative spontaneous platelet aggregation was similar in both groups. Platelet sequestration in oxygenators and arterial line filters was assessed using Indium labelled patients’ platelets reinjected immediately after commencing Iloprost infusion. Platelet sequestration was significantly greater in the placebo group than in the Iloprost group both in the oxygenators (placebo 9.2%, Iloprost 3.4% : P<.001) and in the arterial line filters (placebo 4.8%, Iloprost 0.6% : PC.05).

Blood pressure was significantly lower in the Iloprost group than in the placebo group throughout the infusion period.

Constant infusion of Iloprost during CPB is associated with significantly less platelet sequestration. The importance of this is not only in the conservation of platelet numbers but also the potential reduction in risk of platelet aggregate embolisation.
THROMBOSPONDIN ADSORPTION AND PLATELET ADHESION TO SURFACES
Authors: Adams G A , Hallée C
Published: August 2018

Recent research into cell adhesion has focused on a tripeptide sequence arg-gly-asp (RGD) that is common to a number of cytoadhesive proteins such as von Willebrand factor, fibronectin and fibrinogen. We have previously reported that thrombospondin (TSP) inhibited platelet adhesion to RGD proteins. On further purification of TSP, the inhibitory activity separated away from the TSP. In this report, we demonstate that TSP adsorbs to surfaces and promotes platelet adhesion and thus may belong to this family of cytoadhesins. TSP was purified by heparin affinity chromatography, ammonium sulfate precipitation and sucrose gradient ultracentrifugation. Final preparations were free of TSP aggregates and gave one band on SDS-PAGE. Washed human platelets were radiolabelled, combined with red blood cells and perfused through protein-coated glass or polyethylene (PE) tubes for 7 mins. at a shear rate of 100 s™1. TSP supported platelet adhesion to the same level as fibrinogen (FG), but less than collagen. Platelets were spread but no thrombi were present on the TSP or FG-coated tubes while thrombi formed on collagen-coated tubes. Levels of adsorption were similar for purified solutions of radioiodinated FG or TSP on both glass and PE surfaces. The competitive interactions between these proteins during adsorption to surfaces indicated at equimolar amounts the TSP and FG both deposited on the surface but as FG:TSP ratio increased FG was preferentially adsorbed. These results indicate that TSP is a cytoadhesive protein for platelets when it is adsorbed to surfaces. Thus, the release of TSP from platelets may promote haemostasis or thrombogenesis.

(supported by Ontario Heart Foundation, Canada)
WHAT IS THE OPTIMAL DOSAGE OF LMW HEPARIN IN THE SC TREATMENT OF DEEP VENOUS THROMBOSIS
Authors: Arnesen K E , Handeland G F , Abildgaard U , Gottschalk P , Stene-Larsen G , Nilsen D W T
Published: August 2018

LMW heparin (LMWH) is better suited for subcutaneous (sc) administration than is UF heparin due to higher bioavailability and slower elimination. Optimal dosage for sc treatment of DVT has not been defined. Our previous study suggested that LMWH should be given in doses according to bodyweight (bw), and that sc injection of 100 anti-Xa U/kg bw/12 hrs might result in therapeutic plasma levels (Holm et al. Haemostasis 16, supl 2,30-37, 1986). This dosage is now being evaluated in an open study including patients with venographically proven DVT. Excluded were patients with pulmonary embolism, thrombosis in the IIiacal vein and females beyond 70 yrs of age. LMWH (Fragmin) is administered sc for at least 5 days. Venography is repeated the last day of treatment and evaluated blindly (Marder score). Compared to the previous study in which doses were given according to age and sex, the present protocol results in more uniform and predictable heparin plasma concentrations : Peak concentration day 2 now ranged 0.40-0.75 U/ml (mean 0.58) (n = ll) as compared to the three-fold wider range (0.261.20 U/ml)(mean 0.57)(n = 29) in the previous study. The 12 hrs plasma heparin profile was determined on day 3. Peak concentrations in the 0.44-0.72 U/ml range were found 2.5-4 hrs after injection. The plasma heparin activity was subtherapeutic (< 0.2 U/ml) the last 3-6 hrs of the 12 hrs period. The heparin activity at the next injection averaged 0.05 U/ml (range 0-0.ll). Day to day variation of peak heparin activity in the individual patient, expressed as CV, ranged 11-22% and there was no heparin accumulation.

Conclusions: SC treatment of DVT with LMWH 100 anti-Xa U/kg bw/12 hrs results in peak plasma heparin activity in the 0.40-0.75 U/ml range. The plasma heparin activity was below therapeutic level 3-6 hrs of the 12 hrs period indicating that a larger dose (250 U/kg bw/ 24 hrs divided into 2 or perhaps 3 injections) is preferable.
DOES LOW-DOSE PERI-OPERATIVE HEPARIN ADMINISTRATION AFFECT MORTALITY FOLLOWING MAJOR ABDOMINAL SURGERY?
Authors: Hedges A R , Parker C J , Kakkar V V
Published: August 2018

Current evidence suggests that peri-operative low-dose heparin administration reduces the post-operative frequency of fatal pulmonary embolism and may also reduce the frequency of fatal myocardial infarction. Evidence is now accumulating that anticoagulants affect the course of malignant disease, in particular the formation of metastases. Malignant cells disseminated during surgery may be responsible for metastasis formation.

The aim of this study was to discover whether administration of peri-operative low-dose heparin had any effect on mortality. A retrospective analysis of 1,232 patients undergoing elective abdominal surgery was performed. 658 patients received no heparin and 574 patients received heparin prophylaxis subcutaneously. The two groups were well matched for age, sex, type of operation performed and distribution of pre-existing disease. The number and causes of death are shown below.

Low-dose peri-operative heparin administration reduces postoperative mortality. This reduction is only partly explained by a reduction in cardiopulmonary cases, more significantly there Is a reduction in death due to disseminated malignancy.

A prospective study is planned in patients undergoing operations for malignancy to confirm this finding.
MODIFICATION OF GLUTAMIC AND ASPARTIC ACID RESIDUES OF PLASMINOGEN INHIBITS ITS ABILITY TO FORM AN ACTIVE PLASMINOGEN-STREPTOKINASE COMPLEX
Authors: Holloway D S , Summaria L , Wohl R C , Caprini J A
Published: August 2018

Plasminogen binds to streptokinase in a 1:1 molar complex that has activity as a plasminogen activator. This function of plasminogen, as a cofactor for streptokinase conversion of plasminogen to plasmin, was studied after treatment of Glu-, Lys-, and Mini-plasminogens with 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide (EDC). Amino acid analysis showed that both aspartic and glutamic acid residues were modified by EDC. Activity of the complex formed between streptokinase and the modified plasminogen was measured using the cfhromogenic substrate H-D-Val-Leu-Lys-pNA. Plasminogen, 2.8 uM, was incubated with 40 mM EDC in 50 mM MES buffer, pH 6.0, at 25°C. At various times while reacting plasminogen with the EDC, aliquots were removed for assay. Plasminogen function was assayed by mixing with a slight molar excess of streptokinase for 1 min at 37 C, followed by reaction with 0.1 M substrate, and absorbancy monitored at 405 nm. Modifications of 20% of the glutamic and aspartic acid residues occurred after treatment of plasminogen with EDC. This resulted in 80 to 90% inhibition of activation in all three types of plasminogen. Glu- and Lys-plasminogens reacted more quickly with the EDC than did Mini-plasminogen, with 50% inhibition occurring after 16 ± 5, 16 ± 4, and 67 ± 13 min reaction time with EDC for Glu-, Lys-, and Mini-plasminogens, respectively. Maximum inhibition of activation occurred within 1 hr reaction with EDC for Glu- and Lys-plasminogens but required 2.5 hr for Mini-plasminogen. The time courses for activation inhibition and the modification of the glutamic and aspartic acids of treated Mini-plasminogen were compared. A significant decrease in activation occurred (52%) concomitant with modification of only one or two glutamic acids, followed on further reaction with EDC by more loss of activatability as more glutamic and aspartic acids were modified. The inability of plasminogen to form an active plasminogen-streptokinase complex after modification with EDC indicates that glutamic and aspartic acid residues are involved in the binding site of plasminogen for streptokinase.
THE POSTNATAL DEVELOPMENT OF THE COAGULATION SYSTEM IN THE PREMATURE INFANT
Authors: Andrew M , Paes B A , Milner R A , Powers P J , Johnston M , Castle V
Published: August 2018

A cohort study was performed to determine the postnatal development of the coagulation system in the “healthy” premature infant. Mothers were approached for consent and a total of 132 premature infants were entered into the study. The group consisted of 64 infants with gestational ages of 34-36 weeks (prem 1) and 68 infants whose gestational age was 33 weeks or less (prem 2). Demographic information and a 2 ml blood sample were obtained on days 1, 5, 30, 90, and 180. Plasma was fractionated and stored at −70°C for batch assaying of the following tests: screening tests, PT, APTT; factor assays (biologic (B)); fibrinogen, II, V, VII, VIII:C, IX, X, XI, XII, prekallikrein, high molecular weight kininogen, XIII (immunologic (I)); inhibitors (I), antithrombin III, aα2-antiplasmin, α2-macroglobulin, α-anti-trypsin, Cl esterase inhibitor, protein C, protein S, and the fibrinolytic system (B); plasminogen. We have previously reported an identical study for 118 full term infants. The large number of premature and full term infants studied at varying time points allowed us to determine the following: 1) coagulation tests vary with the gestational age and postnatal age of the infant; 2) each factor has a unique postnatal pattern of maturation; 3) near adult values are achieved by 6 months of age; 4) premature infants have a more rapid postnatal development of the coagulation system compared to the full term infant; and 5) the range of reference values for two age groups of premature infants has been established for each of the assays. These reference values will provide a basis for future investigation of specific hemorrhagic and thrombotic problems in the newborn infant.
LOW TOTAL PROTEIN S ANTIGEN BUT HIGH PROTEIN S ACTIVITY DUE TO DECREASED C4b-BINDING PROTEIN (C4b-BP) LEVELS IN NEWBORNS
Authors: Schwarz H P , Muntean W
Published: August 2018

Vitamin K-dependent coagulation proteins are known to be decreased in the neonatal period. So far no data have been published on protein S (PS), the vitamin K-dependent cofactor for the antithrombotic enzyme, activated protein C (APC) in this period. We determined, therefore, PS antigen, PS activity and C4b-BP,a regulatory protein of the classical complement pathway to which PS is complexed, in 36 neonates. Total PS antigen in newborns was below the range associated with thromboembolism in patients congenitally deficient in this protein (22±9.6%, mean±SD). None of these infants had clinical or laboratory evidence of thromboembolism or DIC. In contrast to the PS antigen level PS activity measured by the ability of APC to prolong the clotting time of a modified APTT assay using PS-immunodep1eted plasma was significantly higher (77.6±14%, mean±SD, p< 0,001), suggesting a shift in PS to the free form. In fact two dimensional immunoe1ectrophoresis studies revealed the absence of protein S-C4b-BP complexes and only one precipitation indicating free PS was seen in 15 out of the 36 infants. In these 15 neonates C4b-BP was below the limit of detection by sensitive quantitative immunob1otting techniques using monoclonal or polyclonal antibodies. In the remaining 21 infants PS-C4b-BP complexes were detected, but in contrast to adult normal plasma approximately 80% of PS was found in the free form. Mixing experiments with normal human plasma and newborn’s plasma indicate that PS in neonate deficient of C4b-BP can bind normally to C4bp. Absence of C4b-BP did not correlate to gestational age. If an equilibrium distribution of PS between bound and free form regulates the cofactor activity of PS for the anticoagulant and profibrino 1ytic properties of APC in normal adults, our study demonstrates that the absence of PS-C4b-BP complexes in newborns and the presence of free PS only may contribute to the increased bleeding risk of premature infants.
COMPARISON OF INTRAVENOUS ANISOYLATED PLASMINOGEN STREPTOKINASE ACTIVATOR COMPLEX (APSAC) AND INTRACORONARY STREPTOKINASE IN ACUTE MYOCARDIAL INFARCTION
Authors: Anderson J , Rothbard R , Hackworthy R , Sorensen S , Fitzpatrick P , Harder V
Published: August 2018

To compare the reperfusion potential of intravenous anisoy-lated plasminogen streptokinase activator complex (APSAC) a new thrombolytic, and standard therapy with intracoronary streptokinase in acute myocardial infarction (AMI), a randomized multicenter reperfusion trial was performed. Consenting patients with clinical signs of AMI and documented coronary occlusion (flow grade 0 or 1) were randomized to treatment within 6 hours of symptoms (mean, 3.4h) with intravenous APSAC (30 U in 2-4 min) or intracoronary streptokinase (bolus, then 2,000 U/min x 60 min). Reperfusion success was defined as grade 2 or 3 flow at 60 min for intracoronary and 90 min for intravenous therapy. A total of 189 patients (pt) were randomized and 179 pt were evaluable for efficacy. Reperfusion was similar for the two treatments: 52% (49/94) for APSAC and 61% (52/85) for streptokinase (p≤0.2). Success was dependent on initial occlusion grade (p≤0.001): 49% (65/133) for grade 0 (APSAC= 45%, streptokinase=54%), but 78% for grade 1 (APSAC=80%, strepto-kinase=77%). The success of intravenous (APSAC) therapy was also dependent on time to treatment: 59% for ≤4h, versus 36% after 4h (p<0.04). APSAC was well tolerated, the change in mean blood pressure after bolus injection being modest (−11 mmHg). Systemic fibrinolysis was somewhat greater after APSAC than low dose streptokinase: fibrinogen levels averaged 39 ± 4% (SE) of control at 90 minutes after APSAC, versus 64 ± 5% after streptokinase (p<0.01) in the subgroup tested. The rate of bleeding complications was acceptable for both regimens, and other adverse reactions were comparable. Rates of early occlusion were low in both treatment groups, and evolution of ECG and enzymatic indicators were similar. Thus, APSAC provides approximately similar reperfusion results as intracoronary streptokinase, especially when given within 4h, but is easier to administer and is well tolerated.
RANDOMIZED FACTORIAL TRIAL OF HIGH-DOSE INTRAVENOUS STREPTOKINASE, OF ORAL ASPIRIN, AND OF INTRAVENOUS HEPARIN IN ACUTE MYOCARDIAL INFARCTION
Authors: Collins R
Published: August 2018

619 patients with suspected acute myocardial infarction (MI) were randomized to receive either a high-dose short-term intravenous infusion of streptokinase (1.5 MU over one hour) or placebo. In addition, using a “2x2x2 factorial” design, patients were also randomized to receive either oral aspirin (325 mg on alternate days for 28 days) or placebo, and separately randomized to receive either intravenous heparin (1,000 IU/hour for 48 hours) or no heparin. Streptokinase (SK) was associated with a non-significant decrease in hospital mortality (7.7% allocated SI< vs 9.2% allocated placebo) and increase in non-fatal reinfarction (3.6% vs 2.9%). There were significantly more minor adverse events after SK (e.g. hypotension, allergies, bruises or minor bleeds), but no excess of strokes or of anaphylactic shock.Aspirin was associated with fewer reinfarctions (2.9% allocated aspirin vs 3.9% allocated placebo; NS), deaths (6.1% vs 10.5%; 2P<0.04) and strokes (0.3% vs 2.0%; 2P<0.1).Heparin was associated with a decrease in reinfarction (1.9% allocated heparin vs 4.9% allocated no heparin; 2P<0.04), though not in mortality (8.3% vs 8.2%; NS), and with a trend towards more strokes (1.6% vs 0.7%; NS) and more bruising and bleeding (14% vs 11%; NS). To assess reliably the effects of SK and aspirin on major endpoints, several hundred hospitals are now collaborating in a large (about 20,000 patients planned) randomized trial (ISIS-2).
EVIDENCE THAT THE C-TERMINAL HEPARIN BINDING DOMAIN ("HEP II") DOMINATES HEPARIN-FIBRONECTIN INTERACTIONS
Authors: Beneckv M J , Kolvenbach C G , Amrani D L , Mosesson M W
Published: August 2018

The interaction of glycosaminoglycans with plasma fibronectin (PFn) may play a role in the conversion of PFn from an “inert” dimeric circulating form to an “activated” multimeric form deposited on the cell surface or in the extracellular matrix. We carried out a quantitative comparison of heparin affinity for PFn and its proteolytic fragments in order to assess the relative importance of heparin interactions with PFn’s various reported heparin-binding domains. We employed affinity chromatography on PFn-sepharose to prepare a subset of fluorescein-labelled heparin molecules with high affinity for PFn, and confirmed that heparin binding to PFn is very sensitive to ionic strength. This suggests that the PFn-sepharose column selectively binds a fraction of highly sulfated heparin molecules. We quantified PFn-heparin affinity in the fluid-phase by monitoring a fluorescence polarization change that occurred as a consequence of the decrease in the rotational diffusion rate of fluorescently-labelled heparin molecules (13.8 kD) as they became “immobilized” by binding to PFn. Scatchard analysis of the heparin fluorescence polarization data obtained for PFn in Tris-buffered saline yielded a biphasic curve with Kd’s estimated at 5 and 130 nM, respectively A 190 kD thrombin fragment, containing the C-terminal "Hep II" domain but lacking the 29 kD N-terminal “Hep I” domain, yielded a linear plot displaying a single class of heparin-binding sites with a Kd of 130 nM Similar results were obtained for the C-terminal 150 kD Fn fragment which also contained the “Hep II” domain. In contrast, the 29 kD N-terminal “Hep I” Fn fragment bound heparin weakly (Kd =25 μM). The nature of the “high affinity (Kd= 5 nM) heparin binding component is uncertain; it may reflect heparin interaction with soluble multimers present in our PFn preparations Our observations suggest that the Kd=130 nM heparin binding component corresponds to heparin interaction with the C-terminal “Hep II” domain We conclude that the N-terminal “Hep I” domain does not participate significantly in heparin binding to soluble dimeric Fn under physiological conditions, whereas the C-terminal “Hep II” domain dominates such interactions
CHARACTERIZATION OF PLATELETS OF A PATIENT WITH A SELECTIVE DEFECT IN ADP-INDUCED AGGREGATION
Authors: Cattaneo M , Lecchi A , McGregor L J , Randi M A , Mannucci M P , Bianchi A
Published: August 2018

We studied a patient (V.R.) with a mild hemorrhagic disorder and prolonged bleeding time. Platelet aggregation induced by ADP (2-50 uM) in V.R.’s citrated PRP or washed platelets (WP) was greatly impaired; in contrast ADP (0.1-1 uM) induced normal platelet shape change of V.R. platelets. Collagen-induced aggregation was absent at low (1-2 ug/ml) but not at high concentration (10 ug/ml). Thrombin at high (1 U/ml) but not at low concentration (0.05 U/ml) induced normal aggregation of V.R.’s WP. Apyrase added to normal PRP or WP caused defective platelet aggregation similar to that observed for V.R.’s platelets. Chymotrypsin-treated WP from V.R. and from a normal control aggregated to the same extent upon addition of fibrinogen. CIE and two-dimensional non-reduced/reduced SDS-PAGE showed no abnormalities of platelet membrane glycoproteins. 125I-fibrinogen (125I-Fg) binding induced by ADP (10 uM) to V.R.’s platelets was absent. Thrombin(0.05-1 U/ml)-induced binding of 125I-Fg to was 20-30% of normal. Chymotrypsin-treated V.R’s platelets bound normal amounts of 125I-Fg. Addition of apyrase to thrombin—stimulated normal platelets lowered 125I-Fg binding to that of thrombin-stimulated V.R.’s platelets. However, 125I-Fg binding to thrombin—stimulated V.R.’s platelets was not affected by apyrase. Unstimulated V.R.’s WP bound the 125I-labeled monoclonal antibody 7E3 (anti Ilb/IIIa) at the same rate and to the same extent as normal WP. Thrombin (1 U/ml) increased the rate of binding of 125I-7E3 to both V.R.’s and control platelets. In contrast, ADP (10 uM) increased the rate of 125I-7E3 binding to control but not to V.R.’s platelets. C-ADP binding to V.R.’s platelets was normal. Therefore, ADP normally interacts with V.R.’s platelets, but does not induce fibrinogen receptors exposure. Selective defects in the ADP pathway of platelet aggregation have not been previously described.
SULFATIDES AND GLYCOLIPIDS IN PLATELETS AND ENDOTHELIAL CELLS
Authors: Schick K P , Shapiro S , Tuszynski G , Slawek J
Published: August 2018

Sulfatides are sulfated glycolipids which are negatively charged and thought to influence receptor mediated activities. Sulfatides have the capacity to provide a surface for the initiation of in vitro coagulation tests and these acidic lipids represent the potential biological surface for the initiation of the contact and intrinsic systems in vivo. Several sulfatides have been demonstrated in blood platelets. We have investigated sulfatides and other glycolipids in endothelial cells and platelets in order to define the cellular sources for sulfatides that would be available for influencing hemostasis. Endothelial cells were derived from primary cultures of human umbilical veins and human platelets were obtained from freshly-collected blood. Cellular lipids were extracted by the Folch method. Sulfatides and glycolipids were purified by silicic acid chromatography, separated by thin-layer chromatography, and quantitated by the assay of sphingosine. Glycolipids were also analyzed by HPLC. Globoside was found to be the predominant glycolipid in endothelial cells while lactosyl ceramide was the predominant glyco-lipid in platelets. Sulfatides were detected by two approaches: 1) Sulfatide synthesis by the incorporation of [35S]-Sulfate; 2) The specific binding of [125I]-thrombospondin and [125I]-von Willebrand’s factor (vWF) to sulfatides separated by thin-layer chromatography (TLC). Several sulfatides were identified in endothelial cells and platelets by virtue of the incorporation of [35S]-sulfate into glycolipids separated by TLC. [125I]-TSP and [125I]-vWF bound to the glycolipids that had incorporated [35S]-sulfate. [35S]-sulfate was primarily incorporated into sulfated galactosyl ceramide but both cells also synthesized complex glycolipids. TSP and vWF were shown to bind to sulfated galactosyl ceramide, a band that comigrated with glycosyl ceramide as well as with two more complex sulfatides in both cells. However, differences in sulfatide synthesis and binding of TSP to sulfatides were observed in endothelial cells from that in platelets. The study indicates that endothelial cells and platelets contain several sulfatides and thus are potential sources for sulfatides for the initiation of coagulation.
HIGH AFFINITY CALCIUM BINDING TO DOMAINES OF PROTEIN C THAT ARE HOMOLOGUS TO THE EPIDERMAL GROWTH FACTOR
Authors: KÖhlin A , Stenflo J
Published: August 2018

In addition to γ-carboxyglutamic acid (Gla)-dependent calcium binding all of the vitamin K-dependent plasma proteins, except prothrombin, have one or two high affinity calcium binding sites that do not require the Gla residues. A common denominator among these proteins (factors IX, X, protein C, protein Z and protein S) is that they have domaines that are homologus to the epidermal growth factor (EGF) precursor. In factors VII,IX,X, protein C and in protein Z the aminoterminal of two EGF homology regions contain one residue of β-hydroxyaspartic acid (Hya) whereas in protein S the aminoterminal EGF homology region contains Hya and the three following contain one β-hydroxyasparagine residue each.

In an attempt to elucidate the role of the EGF homology regions in the Gla independent calcium binding we have isolated a tryptic fragment (residue 44-138) from the light chain of human protein C. The fragment was isolated using a monoclonal antibody that recognizes a calcium ion stabilized epitope that is expressed both in intact protein C and in protein C lacking the Gla domaine.The antibody bound the isolated EGF homology region in the presence of calcium ions but not in EDTA containing buffer. A calcium ion titration showed half maximal binding at approximately 200 μM Ca2+. The metal ion induced conformational change in the isolated fragment was also studied with affinity purified rabbit antibodies against Gla domainless protein C. Antibodies that bound in the presence of calcium ions and that could be eluted with EDTA recognized the metal ion induced conformational change in the isolated EGF homology domain. Our results suggest that one or both of the EGF homology regions are involved in the Gla-independent high affinity calcium binding in the vitamin K-dependent plasma proteins.
RELATIONSHIP BETWEEN LUPUS ANTICOAGULANTS AND ANTIBODIES TO PHOSPHOLIPID
Authors: Triplett D A , Brant J T , Musgrave K , Orr C A
Published: August 2018

The relationship of lupus anticoagulants (LA's) to antibodies (IgG and IgM) to cardiolipin (CL), phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidic acid (PA), detected by an enzyme linked immunoassay, was examined in a series of 100 patients with well characterized LA's. 73% of patients demonstrated antibodies to one or more phospholipids; 62% were positive for CL, 57% were positive for PS, 57% were positive for PI, and 51% were positive for PA. Of the samples with antibodies to phospholipid, 32% had IgG type antibody only, 16% had IgMonly, and 52% had both IgG and IgM antibodies. Of the 100 patients,19% had a history of thrombosis, 8% had ahistory of spontaneous abortion, and 6% had a history of seizure disorder. These complications occurred in the presence (61%) and absence (39%) of detectable phospholipid antibodies. Drug-related LA's were observed in 34 patients; of these 74% were positivefor antiphospholipid antibodies and 24% hada history of thrombosis.

The distribution of antibody types with drug-related LA's was similarto the overall group, with 40% IgG only, 8%IgM only and 52% both IgG and IgM These findings indicate that IgG and IgM antibodies to phospholipid are not observed in all patients with lupus anticoagulants, that thrombosis does occur in this population in the absence of detectable antibodies to phospholipid and that drug-related LA's are associated with thrombosis. Furthermore, there appeared to be little relationship between the degree of prolongation of the aPTT and the titer of anti-phospholipid antibody.
A GENERAL MECHANISM FOR LUPUS ANTICOAGULANTS
Authors: Pengo V , Heine M J , Thiagarajan P , Shapiro s s
Published: August 2018

Although- a number of observations have implied that lupus anticoagulants have immunologic specificity towards anionic. phospholipids, thereby prolonging phospholipid-dependent coagulation tests, this assumption has been directly demonstrated in only one patient with a monoclonal IgM paraprotein. We have tested the generality of this hypothesis directly by isolating five IgG lupus anticoagulants from patients with lupus-like syndromes and/or thrombosis. IgG lupus anticoagulant fractions were isolated free of other plasma proteins and free of contaminating phospholipid by adsorption to and elution from cardiolipin-cholesterol-dicetylphosphate liposomes , followed by chromatography on protein A-Sepharose. Cardiolipin liposomes, but not phosphatidylcholine liposomes, were capable of removing all, or nearly all, lupus anticoagulant activity from patient plasma. Anticardiolipin and lupus anticoagulant activity were both present in acidic fractions on isoelectric focusing. F(ab’)2 fragments retained lupus anti coagulant activity and bound to cardiolipin in an ELISA assay. The affinity-purified IgG preparations reacted with cardiolipin, phosphatidyl serine , phosphatidylinositol and phosphatidic acid, but not with phosphatidylcholine or phosphatidyl ethanol amine, and inhibited calcium-dependent binding of prothrombin and of factor X to phosphatidy1serine-coated surfaces. These data demonstrate a general mechanism for the action of lupus anticoagulants: antibodies that have immunologic specificity towards anionic phospholipids, thereby blocking the calcium-mediated binding of vitamin K-dependent coagulation factors to coagulation-active phospholipid surfaces.
THROMBIN-SPECIFIC SITESOF FIBRINOGEN IN SMALL CELL CARCINOMA OF THE LUNG
Authors: Zacharski L , Memoli V , Rousseau S
Published: August 2018

Thrombin-generated cleavage sites of human fibrinogen have been identified within the connective tissue stroma adjacent to viable tumor cells in fresh frozen sections of small cell carcinomaof the lung (SCCL) by means of immunohistochemical techniques using mouse monoclonal antibodies (designated alpha and beta) to the N-terminal peptides of the fibrinogen alpha and beta chains(provided by G. Matsueda and E. Haber).Specific connective tissue staining with antibody alpha was diffuse while staining with antibody beta was linear and focal. These results indicate thatthrombin is generated in situ in this tumor type. Previous demonstration of an initiator of coagulation together with coagulation factor intermediates associated with viable SCCL tumor cellsin situ(Cancer Res. 43:3963, 1983; Blood 66 (Suppl.1):329, 1985) is consistent with the hypothesis that the tumorcells themselves are responsible for the local thrombin generation. Because favorable effects of anticoagulant therapy with warfarin in SCCL have been demonstrated previouslyin two randomized clinical trials (J.A.M.A. 145:831, 1981; Proc. Am.Soc. Clin. Oncol. 4:191,1985), we postulate that local tumor cell-induced thrombin formation may contributeto self-regulated progression of SCCL through formation of a supportive, fibrin-rich connective tissue stroma (N. Engl. J. Med. 315:1650, 1986) and/or stimulation of cell proliferation (e.g. E.M.B.0. J. 4: 2927,1985; Proc. Natl. Acad. Sci. U.S.A. 83:976, 1986). These results suggest novel treatment strategies for this particular tumor type and justify efforts to identify other tumortypes in which similarmechanisms exist.
PREVENTING VENOUS THROMBOSIS (VT) WITH HEPARIN ALONE OR WITH HEPARIN/DIHYDROERGOTAMINE AFTER ELECTIVE HIP REPLACEMENT, A DOUBLE-BLIND, RANDOMISED, VENOGRAM END-POINT COMPARISON
Authors: Cade J F , Mills K W , Gallus A S , Murphy W
Published: August 2018

Dihydro-ergotaraine (DHE) appears to be synergistic with small doses of hepari when used to prevent VT after general surgery. However, doubt remains whether DHEhas this effect in patients with elective hip replacement (THR). We have therefore compared the results of VT prophylaxis using sub-cutaneous (sc) low-dose heparinalone or sc heparin plus sc DHE in a double-blind, randomised, study of 126 patientshaving elective THR, 98 at centre (1)and 28 at centre (2).

All received 5000 iu sodium heparin, hourly for 7 days, starting 2 hours before surgery at centre (1), or immediately after surgery at centre (2). Patients alsoreceived a separate 0.5 ml (0.5 mg) DHEorplacebo injection each time they receivedheparin. Patients had bilateral ascendingvenography on the 7th postoperativeday, and venograms were read before the treatment code was broken.

These results do not support the presence of synergism between heparin and DHE in this situation.
PREVENTION OF DEEP VEIN THROMBOSIS (DVT) BY ENOXAPARINE (LOVENOXR) AFTER SURGERY FOR FRACTURE OF FEMORAL NECK. ONE DAILY INJECTION OF 40 MG VERSUS TWO DAILY INJECTIONS OF 20 MG
Authors: BARSOTTI J , DABO B , ANDREU J , ALISON D , LEROY J , DELAHOUSSE B
Published: August 2018

In a previous open study, a low-molecular-weight-heparinEnoxa-parine in a dose of 40 mg/24 hrs by subcutaneous (SC) injection, had been shown to be efficient and safe in preventing DVT after total hip replacement (THR), for a non traumatic hip disease. 103 patients (mean age : 82 years ± 10, mean weight : 52 kg ± 10) undergoing an orthopedic operation for fracture of femoral neck were included ina randomized, double blind study. These patients receivedSC Enoxaparine according to two different regimens : 54 patients received the treatment A (2 daily injections of 20 mg), 49 patients received the treatment B (1 daily injection of 40 mg).In both groups, administration of 40 mg of Enoxaparine was begun 12 hours before operation.Patients weretreated for 10-15 days, until bilateral ascending phlebography (BAP) had been completed.

Lower limbs BAP were performed in 97 patients. The incidence of DVT is low and not significantly different betweenthe two regimens :a proximal DVT was detected in 6 patients of the group A and in 2 patients of the group B. This differenceis not significant(p = 0.28).3 patients of each group hada distal DVT. No clinical pulmonary embolism occurred.There was no serious bleeding complication, and the two groups are not significantly different on this point; 2 patients in each group had an important hematoma of the thigh.One hematoma, in a patient who received the treatment B, required a surgical treatment.Red cell transfusion requirementswere 2.6U+1.8 in the group A and 2.5 U ±1.4 in the group B (p = 0.84)There was no significant difference in the daily hemoglobinlevels between the two groups.

This study shows that one daily SC injection of 40 mg of Enoxaparine is as efficient as two daily SC injections of20 mg of Enoxaparine in preventing DVT, in very elderly patients undergoing orthopedic operation for fracture of the femoral neck. The frequencies of bleeding complications in each group were not significantly different.

Enoxaparine (LOVENOXR) - PHARMUKA S.F.
TOTAL HIP REPLACEMENT AND DEEP VEIN THROMBOSIS - RELATIONSHIP TO THE FIBRINOLYTIC SYSTEM
Authors: Eriksson B , Eriksson E , Gyzander E , Teger-Nilsson A C , Riseberg B
Published: August 2018

Reduced fibrinolytic activity increases the risk of recurrent thromboembolism and it has been suggested thatit also plays a role in postoperative thrombosis.

Material and methods. Fibrinolytic parameters were analysed in 29 patients submitted to total hip replacement. Dextran 70 was given as thrombosis prophylaxis. Blood samples weretaken pre operatively, one day and oneweek postoperatively. Venous occlusion test was done in all patients.125 I-fibrinogen test was used for deep vein thrombosis (DVT) screening.Positive test was confirmed with phlebography. Fibrinolytic activity was measured on fibrin plates. Tissueplasminogen activator (t-PA) and itsspecific inhibitor (PAI) were analysed with photometric and immunologicalmethods.

Results. The first postoperative dayt-PA activity decreased and PAI increased significantly. One week after operation only PAI showed significatdifference from preoperative values.10 of the patients developed DVT.The PAI level significantly higher in DVT patients preoperatively. Thisdifference in PAI level was significant compared with non-DVT patients also one day and one week postoperatively.

Conclusion. The recently discovered t-PA inhibitor (PAI) seems to be correlated to postoperative thrombosis in total hip surgery.