bluuebunny/crossref_metadata_2025 huggingface dataset:
An Anti-Tissue Factor Monoclonal Antibody which Inhibits TF Vlla Complex Is a Potent Anticoagulant in Plasma
Authors: Ruf Wolfram , Edgington Thomas S
Published: July 2018

Tissue factor (TF) functions as the receptor and cofactor for factor VIIa (VIIa) to form a proteolytically active TFVIIa complex on cell surfaces. We here demonstrate that most MAbs against human TF were poor inhibitors of TF function in plasma and that they inhibited preformed TF-VIIa complex at a slow rate which was dependent on dissociation of VIIa from the cell surface TF. An exception was defined by one MAb (TF8-5G9) which was an effective immediate anticoagulant in plasma. Binding of TF8-5G9 to TF-VIIa inhibited catalytic function prior to dissociation of the TF-VIIa complex. This analysis thus establishes two distinct mechanisms by which MAbs interfere with TF function. The MAb TF8-5G9 introduces a therapeutic principle for rapid arrest of inappropriate triggering of coagulation by TF as well as the TF-VIIa complex in vivo.
Amidolytic Detection of Prothrombin Activation Products after SDS-Gel Electrophoresis
Authors: Tans Guido , Janssen-Claessen Truus , Rosing Jan
Published: July 2018

In this paper we report a method via which enzymatically active products formed during prothrombin activation can be detected by simple photographic means after SDS-gel electrophoresis, blotting onto a nitrocellulose membrane and visualization with the chromogenic substrate, S2238. After amidolytic detection the same nitrocellulose membrane can also be used for immunologic detection of prothrombin activation products, thus allowing a complete description of product formation during prothrombin activation.

The detection limit of the so-called “amidoblot” is approximately 3 ng thrombin per gel sample which is comparable to the sensitivity of immunoblotting.

It is further shown that the amidoblot technique can also be applied to other coagulation factors for which a suitable chromogenic substrate is available (factor XIIa, kallikrein, factor XIa, factor Xa, plasmin and activated protein C).
Protein Synthesis Inhibition by Cycloheximide Does Not Affect the Acute Release of Tissue-Type Plasminogen Activator
Authors: Tranquille N , Emeis J J
Published: July 2018

The acute release of tissue-type plasminogen activator (t-PA) was studied in perfused rat hindlegs. Pretreatment of rats with the protein synthesis inhibitor cycloheximide (2 mg/kg) at 1, 3 or 5 h prior to perfusion of rat hindlegs did not influence the amount of t-PA released by platelet-activating factor (20 nM) or bradykinin (1 μM). The amount of t-PA activity that could be extracted from hindleg skeletal muscle was not decreased by cycloheximide pretreatment though it was decreased in lung extracts. The in vivo release of t-PA was not affected by cycloheximide pre treatment. The data suggest that the acute release of t-PA from vascular endothelial cells does not require ongoing protein synthesis, but that acutely released t-PA is derived from a stable endothelial storage pool.
Potentiation by Lys-Plasminogen of Clot Lysis by Single or Two Chain Urokinase-Type Plasminogen Activator or Tissue-Type Plasminogen Activator
Authors: Watahiki Y , Scully M F , Ellis V , Kakkar V V
Published: July 2018

Study has been made of the influence of addition of human NH2 terminal glutamic acid plasminogen (Glu-Plg) or human NH2 terminal lysine plasminogen (Lys-Plg) to normal citrated plasma upon the rate of lysis of fully crosslinked plasma clots in the presence of single or two chain urokinase type plasminogen activator (scu-PA/tcu-PA) or tissue plasminogen activator (t-PA). The Specificity of any thrombolytic property was evaluated by measurement of plasma fibrinogen levels. Lys-plgadded to a concentration of 20% of normal plasma plasminogen caused 5 to 6 fold increase in the extent of lysis observed at 6 hours by 100 units/ml of scu-PA and with a small increase in fibrinogenolysis. Glu-Plg added at 20% of normal level had no influence on thrombolysis but at 50% of normal caused increased thrombolysis with rapid depletion of plasma fibrinogen. An apparently synergistic effect of addition of tcu-PA on scu-PA activity was increased by addition of plasminogen (e.g. addition of 20% Lys-Plg increased the lysis rate 4 to 5 fold over the first hour equivalent to an increase of potency of approximately three to four fold). Addition of plasminogen up to double the normal plasma concentration was observed to have no influence on clot lysis in the presence of t-PA. Plasminogen potentiated the rate of lysis by scu-Pr/t-PA synergic mixtures with an approximately 1.5 to 1.9 fold increase in potency. Potentiation occurred without increase in the depletion of plasma fibrinogen. It is concluded that Lys-Plg may be a suitable agent to improve the thrombolytic efficacy of scu-PA, scu-PA/tcu-PA or scu-PA/t-PA therapeutic regimen.
Isolation of Multiple Types of Plasminogen Activator Inhibitors from Vascular Smooth Muscle Cells
Authors: Laug Walter E , Aebersold Ruedi , Jong Ambrose , Rideout Willian , Bergman Barbara L , Baker Joffre
Published: July 2018

Large arteries have a natural resistance to tumor cell invasion thought to be due to the production of protease inhibitors. Vascular smooth muscle cells (VSMC) representing the major cellular part of arteries were isolated from human aortas and grown in tissue culture. These cells were found to produce large amounts of inhibitors of plasminogen activators (PA). Fractionation of VSMC-conditioned medium by heparin-affigel chromatography separated three immunologically and functionally distinct PA inhibitors (PAI), namely PAI-1, PAI-2 and protease-nexin I. The three inhibitors were characterized by functional assays and immunoblotting. PA inhibitor 2 (PAI-2) had little affinity for heparin, whereas PA inhibitor 1 (PAI-l) bound to heparin and was eluted from the column at NaCl concentrations of 0. 1 to 0.35 M. Protease-nexin I, eluted at NaCl concentrations of 0.5 M and higher. Most of the PAI-1 was present in the latent, inactive form. PAI-1 was further purified by ion exchange chromatography on a Mono-Q column. Partial sequencing of the purified PAI-1 confirmed its nature by matching completely with the sequence deduced from the cDNA nucleotide sequence of endothelial cell PAI-1. Thus, human VSMC produce all three presently known PAI and these can be separated in a single heparin affinity purification step.
Ridogrel, a Combined Thromboxane Synthase Inhibitor and Receptor Blocker, Decreases Elevated Plasma β-Thromboglobulin Levels in Patients with Documented Peripheral Arterial Disease
Authors: Hoet B , Arnout J , Van Geet C , Deckmyn H , Verhaeghe R , Vermylen J
Published: July 2018

The combination of thromboxane synthase inhibition with thromboxane receptor antagonism has been shown to result in a strong inhibition of platelet aggregation and a prolongation of the bleeding time (Gresele et al., J. Clin Invest 1987;80: 1435–45).

Ridogrel is a single molecule that efficiently achieves both inhibitions in human volunteers. The present study was performed in patients with obstructive peripheral arterial disease and elevated plasma β-thromboglobulin levels. Patients were treated with either 2 × 300 mg ridogrel or 2 × 300 mg placebo per day for 2½ days, according to a double blind randomised parallel design. Plasma β-thromboglobulin decreased significantly throughout active treatment starting within 2 h after administration; serum and urinary immunoreactive TxB2 levels and urinary 11-dehydro- TxB2 excretion were significantly lower and serum PGE2 and 6-keto-PcF1α levels significantly higher with ridogrel; no changes were observed in the placebo-treated group.

In conclusion this study demonstrates a reduction of platelet activation in vivo by ridogrel.
Effect of Ridogrel on Vascular Contractions Gaused by Vasoactive Substances Released during Platelet Activation
Authors: Janssens W J , Cools F J S , Hoskens L A M , Van Nueten J M
Published: July 2018

Ridogrel (6.3 × 10−6 to 10−4 M) inhibited contractions of isolated rat caudal arteries and rabbit femoral arteries caused by U-46619. The slope of an Arunlakshana-Schild plot (pA2-value: 3.4 × 10−6 M) on the caudal artery was slightly higher than one (1.14). This effect was maximal within}D min of incubation of the blood vessel with the compound and easily reversible. Ridogrel antagonised contractions of isolated rabbit femoral arteries caused by prostaglandin Fzo2α in the same concentration range. Ridogrel also inhibited contractions induced by aggregating rat platelets on isolated rat caudal arteries (itt the presence of ketanserin 4 × 10−7 M) and on isolated rabbit pulmonary and femoral arteries (in the absence of ketanserin). Ridogrel had no effect on Ca2+-induced contractions in depolarised isolated rabbit femoral arteries, and at 10−4 M antagonised serotonin-induced contractions in this blood vessel. Its effect on serotonin-induced contractions was statistically significant but very small on isolated rat caudal arteries. These observations indicate that ridogrel is an antagonist of prostaglandin endoperoxide/thromboxane A2 and prostaglandin F2α raCeptors on vascular smooth muscle.
Bispecific Monoclonal Antibodies Produced by Somatic Cell Fusion Increase the Potency of Tissue Plasminogen Activator
Authors: Branscomb Elizabeth E , Runge Marschall S , Savard Christopher E , Adams Keith M , Matsueda Gary R , Haber Edgar
Published: July 2018

Bispecific monoclonal antibodies that bind simultaneously to human fibrin and tissue plasminogen activator (tPA) enhance the fibrinolytic potency of tPA. Two bispecific antibodies (F36.23 and F32.1) were generated by somatic cell fusion. Antibody F36.23 derives its tPA binding from monoclonal anti-tPA antibody TCL8 and its fibrin binding from monoclonal antifibrin antibody 59D8. After purification from cell supernatants and ascites by two steps of affinity chromatography, hybrid-hybridoma bispecific antibody F36.23 simultaneously bound tPA and fibrin in solution and in solid-phase assays. In an assay for the lysis of human fibrin monomer, F36.23 increased the fibrinolytic potency of tPA by 5 to 10 fold, regardless of whether the bispecific antibody had been combined with the tPA before or during the assay. Bispecific F36.23 F(ab′)2 also bound tPA and fibrin simultaneously, and the enhancement in fibrinolysis in the presence of F36.23 F(ab′)2 was identical to that in the presence of intact F36.23. The second bispecific antibody, F32.1, was produced by an alternative strategy that has a wider potential for applicaton in other systems. Hybridoma bispecific antibody F32.1 was derived from the fusion of immune splenocytes (in mice immunized with a synthetic oligopeptide representing the amino terminus of the α-chain of human fibrin) with the anti-tPA cell line TCL8. The properties of hybridoma bispecific antibody F32.1 and its F(ab′)2 were indistinguishable from those of hybrid-hybridoma bispecific antibody F36.23 in solid-phase binding assays and in assays of fibrinolysis. Bispecific antibodies produced by somatic cell fusion, particularly in the form of F(ab′)2, may have potential for use in clinical thrombolysis.
Plasmin inactivation in Plasma
Authors: Haselager E. M , Goote Th. M , Vreeken J
Published: July 2018

Inhibition and inactivation of plasmin is ascribed to α2-macroglobulin, α1-antitrypsin and c1-esterase inhibitor. In an “overall” inactivation test of plasmin in plasma, which comes perhaps closest to rapid inactivation of plasmin in the circulating blood, we only found a correlation between the immunological concentration of α2-macroglobulin and the plasmin-inac-tivating capacity of the plasma, but no correlation with the immunological concentration of the other inhibitors mentioned. Therefore, α2-macroglobulin seems to be the most important plasmin inhibitor in relation to thrombosis.
Monoclonal Purified F VIII for Continuous Infusion: Stability, Microbiological Safety and Clinical Experience
Authors: Schulman S , Varon D , Keller N , Gitel S , Martinowitz U
Published: July 2018

Replacement therapy for patients with hemophilia A postoperatively or for major hemorrhage, administered as a continuous infusion, is efficient and reduces the requirement for factor VIII (F VIII). The convenience of the method is increased by using a minipump and not diluting the concentrate further after reconstitution. A monoclonally purified F VIII concentrate (Monoclate-P®), was evaluated for its stability after reconstitution in different infusion systems, for its microbiological safety as well as clinical safety and efficacy in continuous infusion. The F VIII activity was unaffected by 2 of the 3 infusion systems at room temperature during 15 days, whereas in the third (CADD®-1) it decreased below 80% of initial value after 3-7 days. Addition of heparin (1 U/ml) or low molecular weight heparin (1 anti-Xa U/ml), which are used to prevent thrombophlebitis at the site of infusion, did not affect the stability. Nine out of 9 samples taken from the infusion systems after 3 days and again after 7 days were sterile. After inoculation with Staphylococcus aureus or Escherichia coli the bacterial growth in samples of the reconstitued concentrate was not different from that in lidocain in saline or heparin in saline. F VIII was given in continuous infusion with a minipump (Infu-Med™) to 12 patients undergoing major surgery and 8 patients with major hemorrhage for a total of 157 days. A progressive decrease of the clearance was seen during the first 5 days of infusion from 3.0 to 1.7 ml/kg/h. Hemostasis was effectively achieved, and no infectious complications were registered. The administration of monoclonally purified F VIII with a minipump is safe, efficient and convenient and reduces the consumption of F VIII when high doses are needed. Some patients can be discharged earlier and admission to the hospital may even be unnecessary, since high and steady F VIII levels can be maintained.
Thromboplastin Related Differences in the Determination of International Normalised Ratio: A Cause for Concern?
Authors: Kitchen S , Walker I D , Woods T A L , Preston F E
Published: July 2018

When the International Normalised Ratio (INR) is used for control of oral anticoagulant therapy the same result should be obtained irrespective of the laboratory reagent used. However, in the UK National External Quality Assessment Scheme (NEQAS) for Blood Coagulation INRs determined using different reagents have been significantly different.

For 18 NEQAS samples Manchester Reagent (MR) was associated with significantly lower INRs than those obtained using Diagen Activated (DA, p = 0.0004) or Instrumentation Laboratory PT-Fib HS (IL, p = 0.0001). Mean INRs for this group were 3.15, 3.61, and 3.65 for MR, DA, and IL respectively. For 61 fresh samples from warfarin-ised patients with INRs of greater than 3.0 the relationship between thromboplastins in respect of INR was similar to that observed for NEQAS data. Thus INRs obtained with MR were significantly lower than with DA or IL (p <0.0001). Mean INRs for this group were 4.01, 4.40, and 4.59 for MR, DA, and IL respectively.

We conclude that the differences between INRs measured with the thromboplastins studied here are sufficiently great to influence patient management through warfarin dosage schedules, particularly in the upper therapeutic range of INR. There is clearly a need to address the issues responsible for the observed discrepancies.
Relation between Plasminogen Activator Inhibitor-1 and Hepatic Enzyme Concentrations in Hyperlipidemic Patients
Authors: Bruckert E , Ankri A , Glral P , Turpin G
Published: July 2018

Plasminogen activator inhibitor type-1 (PAI-1) is a key determinant of the fibrinolytic capacity. Its activity correlates with most of the characteristic features of insulin resistance syndrome, i. e. obesity, high blood pressure and hyperlipidemia.

We measured plasma PAI-1 antigen levels in 131 asymptomatic men (aged 44.2 ± 11 years) who had been referred for hyperlipidemia. Those taking medication and those with a secondary hyperlipidemia were excluded.

We confirmed the correlation between PAI-1 levels and the following variables: body mass index, blood pressure, triglyceride concentration, and blood glucose and insulin levels before and after an oral glucose tolerance test. We also found a significant and independent correlation between PAI-1 and the concentration of the hepatic enzymes glutamyl transferase, alanine aminotransferase and aspartate aminotransferase.

Mild liver abnormalities (presumably steatosis) may thus be one of the factors accounting for high plasma PAI-1 levels in hyperlipidemic patients.
Proteolytic Inhibitors, Contact and Other Variables in the Release Reaction of Human Platelets
Authors: Zucker Marjorie B.
Published: July 2018

Platelet-rich plasma (PRP) containing platelets labelled with 14C-serotonin was used to characterize the release reaction. Heparin promotes release, and the reduced release response to epinephrine observed in heparinized compared to citrated PRP is probably due to the earlier reaction. The release reaction can occur in citrated PRP at pH 7.0, is marked between pH 7.2 and 7.6, and decreases above pH 8.0. It is retained better when PRP is stored below pH 7.7. Adenosine, prostaglandin E1, and theophylline inhibit both aggregation and release, probably because they elevate cyclic AMP. D- and L-tosyl-phenylalanine chloromethylketone (TPCK) also inhibit both aggregation and release at a concentration of 0.1 mM whereas 0.1 mM D- and l-L-chloro-2-tosylamido-7-amino-2-heptanone (TLCK) and 5 mM diisopropylfluoro-phosphate (DFP), like aspirin, inhibit only release. Colchicine (0.1 mM) alters platelet shape but does not affect release or aggregation. Centrifugation of PRP at 37° C causes release which responds to inhibitors and pH changes in a manner similar to release induced by aggregation with ADP or epinephrine. Release occurs in thromb-asthenic platelets centrifuged at 37° C and is increased by epinephrine. Release of ATP and ADP correlates significantly with release of 14C-serotonin. Thrombin plus CaCl2 causes release of β-glucuronidase but not of zinc.
Kymographic Recording of Clot Retraction: Effects of Papaverine. Theophylline and Cytochalasin B
Authors: Majno G , Bouvier C.A , Gabbiani G , Ryan G.B , Statkov P
Published: July 2018

Clot retraction was studied by setting up ribbon-shaped clots in a pharmacological bath and recording the progress of contraction on a kymograph. Drugs could be added to the bath or washed out at any stage. Papaverine, theophylline and cytochalasin B each reversed the upward course of the kymograph tracing ; contraction began again when the drugs were washed from the bath. This phenomenon resembles the behaviour of smooth muscle under similar circumstances.
Blood Coagulation and Fibrinolysis in a Random Sample of 788 Men 54 Years Old
Authors: Korsan-Bengtsen Kristoffer , Wilhelmsen Lars , Tibblin Gösta
Published: July 2018

In a population of 788 men 54 years of age plasma clotting times, factor II-VII-X activity, factor VIII, fibrinogen, fibrinolytic activity and plasminogen have been compared to anthropomorphic variables, blood lipids, blood sugar, blood pressure, heart rate, lung function and smoking.

It was found by multiple regression analyses that fibrinogen, plasminogen and factor II-VII-X activity were correlated to serum cholesterol, and secondary to systolic blood pressure, heart rate and blood sugar.

Independent of these correlations, fibrinogen, plasminogen and factor II-VII-X activity were also negatively correlated to FEV1 and VC which are measures of lung function.

Smokers have a higher fibrinogen level than non smokers and this is not due to the decreased lung function in smokers. PTT (partial thromboplastin time) carried out in silicone tubes is correlated to serum cholesterol.

There is a negative correlation between fibrinolytic activity and weight, subscapular and thorax skinfolds, chest and waist chcumference indicating that obese subjects have a low fibrinolytic activity. There is also a negative correlation between fibrinolytic activity and serum triglycerides but this is insignificant when the effect of obesity is taken into account.
Influence of Platelet Count on Haemostatic Plug Formation and Plug Stability
Authors: Bergqvist D , Arfors K.-E
Published: July 2018

The initial haemostatic process in thrombocytopenic animals after transection of arterioles and venules was investigated in a rabbit mesenteric preparation. Graded thrombocytopenia with a bone marrow suppressant (busulphan) and anti-platelet serum gave comparable results. With decreasing platelet counts there was progressive prolongation in haemostatic plug formation time, and a significant non-linear relation between platelet count and haemostatic plug formation time. Haemostatic plugs formed under thrombocytopenic conditions were more stable than normal. The difference in stability normally seen between plugs in arterioles and venules disappeared with decreasing platelet counts. The relationship between venular and arteriolar haemostatic plug formation time was also reduced with decreasing platelet counts.
Oral Anticoagulant Therapy and its Control: An International Survey
Authors: Lam-Po-Tang P. R. L. C , Poller L
Published: July 2018

A survey for the International Study Group for Anticoagulant Control has been conducted in which a questionnaire was sent to members in 39 countries. Information based on 37 replies obtained indicated that the most widely used test in oral anticoagulant therapy is the Quick one stage prothrombin estimation. There is a great diversity in the method of reporting prothrombin time results. Percentage activity of normal on the basis of saline dilution curves is the most popular. Only six countries have national or regional systems for anticoagulant control.

Recommendations for the therapeutic range of dosage vary widely. The upper limit in one country may be the lower limit in another. In many centres doses of anticoagulant drugs are advocated which elsewhere are considered either inadequate anticoagulation or dangerous overdosage. The variation of therapeutic range may still be considerable even when a standard thromboplastin is available.

Data collected from the survey indicates the need for urgent measures to provide a uniform basis of laboratory control of clinical dosage of anticoagulant drugs. Until such measures are implemented it is meaningless to try to compare the efficacy of oral anticoagulant therapy from country to country or even to attempt multicentre trials within national boundaries.
An International Collaborative Study to Investigate Standardisation of Hirudin Potency
Authors: Longstaff Colin , Wong Man-Yu , Gaffney Patrick J
Published: July 2018

An international collaborative study has been carried out to investigate the reproducibility of hirudin assays in 13 laboratories using four recombinant hirudins and one natural, sulphated product. A simple assay procedure was proposed involving the titration of α-thrombin with inhibitor and measurement of residual activity using a chromogenic substrate. A standard α-thrombin preparation was supplied to ensure that this reagent was of uniform quality throughout the study. The method appeared to present no difficulties and laboratories reported similar potencies for the 5 hirudin samples, in line with expected values. This gave 200–222 Thrombin Inhibitory Units/ampoule (TIU/ampoule) of lyophilised hirudin, with geometric coefficient of variation (gcv) values ranging from 10.15–15.97%. This corresponds to specific activities of approximately 14,300–15,900 TIU/mg protein. This is close to the upper limit of previously reported values of specific activity. We conclude that the precision of this determination compared with the wider range of values in the literature (8,000–16,000 thrombin inhibitory units [TIU]/mg) results from the use of good quality standard α-thrombin by all laboratories. This study has important implications for hirudin standardisation.
A Novel Polymorphism in Glycoprotein IV (Replacement of Proline-90 by Serine) Predominates in Subjects with Platelet GPIV Deficiency
Authors: Kashiwagi Hirokazu , Honda Shigenori , Tomiyama Yoshiaki , Mizutani Hajime , Take Hironori , Honda Yumiko , Kosugi Satoru , Kanayama Yoshio , Kurata Yoshiyuki , Matsuzawa Yuji
Published: July 2018

To clarify the molecular basis of the deficiency of glycoprotein IV (GPIV) of the platelet surface, we analyzed GPIV cDNA synthesized from platelet RNA of five unrelated Japanese subjects whose platelets did not express GPIV.

We confirmed the presence of normal-sized GPIV mRNA in platelets from subjects with GPIV deficiency. The sequence of platelet GPIV cDNA from GPIV deficient subject showed three differences when compared with the published sequence; 1) a replacement of a 478CCT codon for proline-90 by TCT for serine, 2) a four-base insertion in the 3′-noncoding region, and 3) a substitution of A for 79C in the 5′-noncoding region. The replacement of Pro90 by Ser predominates in subjects with GPIV deficiency; that is, four out of five platelets with GPIV deficiency contained GPIV mRNA encoding GPIVSer-90, while all platelets from 17 GPIV positive subjects had GPIV mRNA encoding GPIVPro-90. The sequence of platelet GPIV cDNA which did not encode GPIVSer-90 from a subject with GPIV deficiency revealed no abnormality in the coding region. The four-base insertion in the 3′-noncoding region and the substitution of A for 79C in the 5′-noncoding region seems to be unrelated to the expression of GPIV.

The substitution of Ser for Pro90 might alter the GPIV structure or impair GPIV biosynthesis, resulting in a lack of detectable GPIV. This hypothesis remains to be tested.
Beta-Lactam Antibiotics Inhibit Agonist-Stimulated Platelet Calcium Influx
Authors: Burroughs Susan F , Johnson Gerhard J
Published: July 2018

(β-lactam antibiotics cause platelet dysfunction with reversible agonist-receptor inhibition, irreversible [14C]-penicillin binding, and inhibition of agonist-stimulated elevation in cytosolic Ca2+ ([Ca2+]i), occurring after 24 h exposure in vitro and after in vivo treatment. We investigated β-lactam antibiotic-induced inhibition of rises in [Ca2+]i stimulated by thrombin, sodium arachidonate or A23187 to determine whether Ca2+ influx or intracellular release was primarily affected. The mean rise in [Ca2+]i, measured with fura-2-AM, was inhibited 43.7-84.1% by penicillin when the extracellular Ca2+ concentration ([Ca2+]e) was 1 mM, but was significantly less inhibited when [Ca2+]e was <1 μM. NiCl2 (2 mM), that blocks Ca2+ influx, caused inhibition comparable to penicillin. MnCl2 (1 mM), that quenches the intracellular fura-2 signal, significantly decreased the rise in 1 mM [Ca2+]i when [Ca2+]e was 1 mM, but did not increase the inhibition caused by penicillin. Penicillin did not inhibit the rise in [Ca2+]i stimulated by inositol-1,4,5-trisphosphate or GTPγS. Therefore, β-lactam antibiotics inhibit agonist-induced elevations of [Ca2+]i primarily through inhibition of Ca2+ influx, which probably accounts for the irreversible inhibition of platelet function seen after prolonged in vitro or in vivo treatment.
Antithrombin III And Anti-Factor Xa Levels In Two Patients With Acute Promyelocytic Leukemia And Diffuse Intravascular Coagulation Treated With Heparin
Authors: Sandler R M , Liebman H , Patch M J , Teitelbaum A , Levine A , Feinstein D I
Published: July 2018

Serial blood coagulation studies in two patients with acute promyelocytic leukemia (APL) showed them to have disseminated intravascular coagulation (DIC) as evidenced by hypofibrinogenemia, positive plasma protamine sulfate paracoagulation tests and elevated fibrin degradation products. Serial samples were assayed for functional antithrombin- II I (AT-III) and anti-factor Xa (anti-Xa) activity before, during, and after heparin therapy. Heparin was detected by a modified thrombin time. AT-III was determined by fluorometric measurement of residual thrombin activity on a synthetic substrate (“Protopath” System, Dade Corporation, Florida, U.S.A.). Anti-Xa was determined by coagulation assay of residual Xa after prolonged incubation of prepared Xa with the test plasma. Patient #1 showed normal AT-III levels before and during heparin therapy, which fell to lower levels after the heparin was stopped. Anti-Xa was normal initially and then rose to very high levels during heparin therapy. There was an abrupt fall on cessation of heparin. Patient #2 showed normal AT-III levels at all times, but anti-Xa levels were initially low, rising to normal or high levels only when heparin was detectable in the plasma. It is concluded that neither DIC nor heparin necessarily cause a low AT-III activity in APL, and that therapeutic AT-III replacement may not be necessary. The anti-coagulant effect of heparin, as evidenced by a rise in the anti-Xa level, appears to be normal.
Heparin Administration And Precision Protamine Reversal In Coronary Bypass Surgery
Authors: Markham Charles
Published: July 2018

A two-tiered testing procedure was developed which uses the activated clotting time (ACT) dose-response curve for the initialization and maintenance of heparinization while using the whole blood activated recalcification time (wBART) for a more precise control of protamine neutralization. The wBART, the most sensitive test for heparin induced hypocoagulability, parallels the ACT and is prolonged by protamine excess, allowing a rapid and accurate titration procedure. This twotiered test protocol results in decreased protamine usage and less blood loss compared to published data in a comparable patient population.

The protocol was applied to a study with 55 by-pass patients to compare two commercial beef lung heparins with respect to anticoagulant effectiveness, intraoperative ACT levels, total heparin usage, post-operative blood loss and protamine requirements. Platelet counts were also monitored before and following surgery. No statistically significant differences were observed between the two heparin products in any of these parameters. Details of the two-tiered protocol and the beef lung heparin study will be presented.
Urokinase-Related Fibrinolytic Activity In Human Plasma
Authors: Wijngaards G , Kluft C
Published: July 2018

Blood represents a complex system containing plasminogenn activators. At present, three different types of plasminogen activator activity are distinguished. It has been assumed that urokinase is not one of the blood activators, but recently an improbably high concentration of urokinase antigenic
material has been claimed to be present in plasma.

The present study was performed to establish if urokinase activity participates in blood fibrinolysis. This was investigated by measuring quenching by antibodies. The dextran sulphate euglobulin fraction (DEF) of plasma was prepared and its fibrinolytic activity was measured in a clot lysis time method with and without addition of various anti-urokinase immunoglobulin fractions (AUK’s).

Considerable quenching of the DEF activity by the AUK’s was found. The quenchings reached maximum values at high antibody concentrations. The quenching curves of the fibrinolytic activity of both urokinase and DEF could be superimposed for each AUK. This quenchable activity was also very sensitive to inhibition by a specific urokinase inhibitor from human placenta. Addition of kaolin to DEF, to include factor XII-dependent fibrinolysis, showed an increase of the fibrinolytic activity which was not quenched by AUK. Double diffusion analyses did not result in the demonstration of urokinase antigen in plasma. This was in agreement with the amount of activity quenched (comparable with about 1 ng urokinase/ml plasma) and the detection limit for a visible precipitation line.

These observations indicate that plasma contains urokinase-related activity. This activity is assumed to belong to the factor XII-independent proactivator system.
Rapid Analytical Techniques For Measuring The High And Low Molecular Weight Contents Of Urokinase Samples
Authors: Devine E M , Shen L L , Saldivar A C , Gendrich R L , Wiegand R G , Holleman W H
Published: July 2018

Two assay procedures have been developed for measuring the ratio of the two molecular weight forms of urokinase commonly found in either urine or the fluid of cultured human kidney cells. The first method is a mini gel filtration technique which rapidly separates the high molecular weight (HMW) and low molecular weight (LMW) urokinases. This procedure can be used for urokinase samples of any purity. The biological active fractions are assayed by the fibrin plate technique. These biological activities are converted to relative molar amounts by the use of the molar activity constant for each molecular weight form. The second procedure is based on the differing responses of the two molecular species in the fibrin plate and the Ploug clot lysis assay systems. The comparison of assay responses of an unknown with a predetermined curve obtained from known mixtures of HMW and LMW urokinases results in a measure of the relative amounts of the two molecular forms. This system is not applicable to samples of low purity.

The two systems gave comparable results. For example, the WHO First International Reference Preparation (66/46) of Urokinase is 31% HMW urokinase by the differential method and 32% HMW urokinase by the gel filtration technique. Both methods require small amounts of sample (400 IU or less) and should replace the use of sodium dodecylsulfate polyacrylamide gel electrophoresis for urokinase molecular species determinations.
Studies Of Mechanism Of The Augmentation Of Streptokinase (SK) Activator By Fibrinogen Or Fibrin
Authors: Camiolo S M
Published: July 2018

The recently reported augmentation of SK activator activity by fibrin(ogen) was found to be retained when lysed fibrin(ogen)(FDP) or gel-filtered FDP pools within the range of 35,000 to 100,000 mol. wt. were used in place of the intact molecules. In all cases, the effect was the result of complexing of protein with a fraction of the SK added which migrated with it as was visualized on flat-bed electrofocusing. Anti-streptokinase-antibody (Kabi) inhibited the formation of an active activator complex species and served to identify an apparently protected, or otherwise activated, streptokinase molecule forming in the presence of fibrin(ogen). Electrofocusing conducted in 6M urea to separate the complexes, showed that SK fragments differed in size and in isoelectric point when SK was preincubated with plasminogen (1:1 mole ratio) in the presence or absence of FDP. A difference in the breakdown pattern of SK was also observed when [125I] SK activator, formed in the presence and absence of fibrin, was examined in SDS gel electrophoresis.
Plasminogen Activator Production By Malignant Human Cells: In Vivo And In Vitro Studies
Authors: Marsh N A , Duffy M J , Gaffney P J
Published: July 2018

A link between malignancy and impaired haemostatic function has been suggested for some time. However, the clinical implication remains confused and mutually exclusive hypotheses concerning the nature of this link are in vogue. This report concerns part of a study designed to establish the nature of this association and to define whether it is causal or accidental.

Initial experiments indicated that there was no clear correlation between malignancy, transformation and plasminogen activator (PA). Indeed, cultures of the malignant cell lines Hela and Hep2 and of a transformed lung fibroblast line produced little PA while two normal lines, MRC 5 and WI38 produced very high amounts of PA. In vivo data were obtained from human breast tumours which had been classified into those which contained steroid binding receptors and those which did not. PA levels were measured in tumour extracts by means of two assays, a functional 125I-labelled fibrin digestion technique and a radioimmunometric method. Enzymic activity was also screened on a range of synthetic chromogenic substrates.

PA levels were consistently lower in those breast tumours containing steroid receptors, the ratio of PA activity in receptor-negative and receptor-positive tumours ranging from 1.43 to 3.03. All samples contained small amounts of antiplasmin-like activity and some contained other haemostatic component activities including thrombin, kallikrein and Factor Xa.

Our results do not confirm the findings of other workers who have demonstrated a positive correlation between breast tumour plasminogen activator activity and the level of steroid receptors. Plasminogen activator levels may thus be of limited prognostic value in determining the hormoneresponsiveness of breast tumours and cast doubt on the view that malignant cells produce increased amounts of PA.
Haemostatic Function And The Prediction Of Fatal Reinfarction
Authors: Haines A P , Howarth D J , North W R S , Meade T W , Millar-Craig M W
Published: July 2018

Haemostatic function was studied in over 500 patients within a day or two of their admission to the Coronary Care Unit at Northwick Park Hospital. All were followed up. Those who died within e year of admission were identified and the cause of death established. This paper deals with early mortality in those with confirmed myocardial infarction. At six weeks, 40 of these patients had died of re-infarction. Mean admission levels of factor VIII (clotting and antigen) and of fibrinogen were, by 15-20%, significantly higher in those who died than in those who survived. Mean fibrinolytic activity was about 50% less. These three measures - factor VIII, fibrinogen and fibrinolytic activity - thus appear to have some predictive value for fatal re-infarction. The differences between those who died and survived might have been the consequence of more extensive initial infarcts in the former. Alternatively, disordered haemostasis tending towards “hypercoagulability” may be of causal significance in fated re-infarction.
Alterations Of Hemostasis During Cardiopulmonary Bypass (CPB): A Comparison Between Membrane And Bubble Oxygenators
Authors: Bick R L , Arbegast N R , Schmalhorst W R
Published: July 2018

Alterations of hemostasis during cardiopulmonary bypass (CPB) using bubble oxygenators have been previously defined and found to consist of a severe platelet function defect, a primary hyperfibrino(geno)lytic syndrome, and minimal thrombocytopenia. This study compares defects in hemostasis with membrane oxygenators and bubble oxygenators. 30 consecutive patients were studied and all patients studied were undergoing elective coronary artery bypass surgery. Tests of hemostasis included thrombin and reptilase times, protamine corrected thrombin times, soluble fibrin monomer, fibrinogen degradation products, fibrinolytic assays, platelet counts, and tests of platelet function. Studies were drawn pre-bypass, mid-bypass, and 1 hour post bypass. It was found that thrombocytopenia was much less in membrane patients. All patients developed a primary hyperfibrino(geno)lytic syndrome and the degree of this was equal in bubble or membrane oxygenators. Platelet dysfunction also was seen in all patients but was significantly different between the two oxygenation systems. At one hour postop, membrane patients showed no correction of platelet function as assessed by adhesion (14%), while those perfused with bubble oxygenators showed significant correction (67%) at one hour postop.

In conclusion, the primary hyperfibrino(geno)lytic syndrome occurring during cardiopulmonary bypass appears to be of equal significance regardless of oxygenation mechanism. Less thrombocytopenia, but more platelet dysfunction is seen with the membrane system.
Free ATP In Blood During Haemorrhage
Authors: Kratzer M A A , Born G V R
Published: July 2018

When blood vessels are injured so that they bleed, circulating platelets adhere to the damaged vessel wall and aggregate within the first seconds. Ihe mechanism of the initial platelet aggregation remains uncertain. To investigate the initiation stage of haemostasis, the carotid arteries of rats were punctured with a 100 μm needle and free ATP, as an indicator of ADP, was measured in the emerging blood.

This was brought into contact with luciferin-luciferase in a polyethylene tube, internal diameter 0.8 mm. The light produced at the blood/enzyme interface was measured with a sensitive photon - counting device which gave backgroundn counts of 1 photoelectron/sec. and could detect < 10-8M ATP in 2 μl blood

When an artery was injured, the emerging blood contained about 10-7 M ATP in a first peak after about 2 sec. After about one min. the ATP concentration rose to a second peak of about 5.10-6M This was decreased by heparin (500 U/kg body weight) or by chlorpromazine (1 mg/kg). The observations suggest that the second peak represents ATP released frem platelets. The source of ATP accounting for the first peak remains uncertain; possibly this ATP is released fran red cells undergoing high shear stress frem the haemodynamic effects of haemorrhage.
Disseminated Intravascular Coagulation Complicating The Perinatal “Dropped-Dead” Syndrome: Lack Of Influence Of Cardio-PulmoNary Resuscitation
Authors: Peliowski A , Rand C , Barr R , Zipursky A
Published: July 2018

A diagnosis of perinatal “dropped-dead” syndrome (PDDS)- defined as an Apgar score of 0 or 1 at one minute after birth - was made on 301/4800 (6%) babies admitted to a neonatal intensive care unit during the period 1973-1980. This disorder may be complicated by clinical features of d is seminated intravascular coagulation (DIC). Laboratory in vestigation of hemostasis was undertaken on 174/301. DIC was defined as a plasma fibrinogen concentration of ⋜150mg per dl. or a 2 unit thrombin time of ⋝ 30 seconds. A retro spective analysis was undertaken in order to determine (a) whether the occurrence of DIC was influenced by the outcome of cardiopulmonary resuscitation (CPR), as reflected in the Apgar score at 5 minutes after birth, and (b) if the features of DIC were transient or persistent. A diagnosis of DIC was made in 69/174 (41%) and it was unrelated to the interval between birth (B) and the time of investigation (I): p = 0.2687. Furthermore, DIC was unrelated to change in the Apgar score (between estimates at one and 5 minutes):t p = 0.3260. Moreover, the change in Apgar score was unrelated to either gestational age (GA): p = 0.0788, or birth weight (BW): p = 0.4776, although the BI interval was related directly to both GA: p = 0.0137 and BW: p = 0.0052. Thus, the outcome of CPR is unrelated to degree of prematurity; but the more premature the baby the sooner he/she is studied after birth. DIC is a common complication of PDDS. It is evident even many hours after birth and is not influenced by the outcome of CPR. The efficacy of alternative therapeutic strategies remains to be evaluated
Disseminated Intravascular Coagulation: A Clinical/Laboratory Correlation In 48 Patients
Authors: Bick R L
Published: July 2018

Disseminated intravascular coagulation (DIC) is a frequent clinical entity spanning from a moderately severe bleeding disorder to a catastrophic, fulminant, and often fatal form usually associated with hemorrhage or, less commonly,as diffuse thromboses. The clinical and laboratory features of DIC remain confusing and controversial. To critically evaluate the usefulness of coagulation tests in aiding in the diagnosis and monitoring of therapy in DIC the clinical and laboratory findings were summarized in 48 patients with DIC. All patients were subjected to a prothrombin time (PT), activated partial thromboplastin time (PTT), reptilase time (RT), thrombin time (TT), fibrin(ogen) degradation products (FDP), platelet count, protamine sulfate test (PSO4), fibrinogen determination, and biological antithrombin-III (AT-III) level at the time of diagnosis. In addition, these same laboratory modalities were used to monitor patients during and after therapy. In this series of 48 patients, 38 patients had acute DIC and 10 patients had chronic DIC. In those patients with acute DIC, 100% of patients presented with hemorrhage and 53% of patients had thrombosis; 26% of patients died of their DIC type syndrome. In those patients with chronic DIC, 100% presented with hemorrhage, 80% presented with thrombosis, and none died of their intravascular clotting process. The probability of a pre-treatment abnormality in acute DIC was: FDP > AT-III = platelet count PS04 > TT > PT > fibrinogen level > PTT > RT. The probability of pre-treatment abnormalties in chronic DIC was: FDP > PSO4 = PT > AT-III = RT platelet count fibrinogen level = TT. These studies suggest the FDP level, the AT-III level, PSO4, and fibrinogen level to be reliable for aiding in the diagnosis of acute DIC. In chronic DIC the fibrinogen level, PS04, PTT, and AT-III level appear to be the most reliable indicies.
Hemostatic Changes After Plasma Exchange Therapy For Fulminant Hepatic Failure
Authors: Takata N , Kusumi S , Fujimura K , Kuramoto A
Published: July 2018

Although it is well known that fulminant hepatic failure (FHF)might be often complicated with disseminated intravascular coagulation (DIC), its bedside diagnosis and control are rather confused. Hemostatic change after replacement therapy might be helpful to distinguish wheather the abnormal clotting tests are owing to the defects of protein synthesis or to excessive consumption. A 38-year-old woman with relapse of acute myeloblastic leukemia and FHF(post-transfusion hepatitis, type of non-A,non-B) received 2 courses of plasma exchange therapy (4 and 3L) using Hemonetics M-30 with 6,000 U/day of heparin sodium continuous i.v. and died on 4th hospital day. Postmortem necropsy revealed massive hepatic necrosis and fibrin thrombi in kidneys. Before the beginning of plasma exchange, results of bedside tests were as follow;platelets 44×109/l(decreased from 240 within a week ), PT over 90", aPTT 26", fibrinogen 120 mg/dl and serum FDP 10 mcg/ml. Serial observations of hemostatic parameters 1, 4, 8 and 12 hours after the end of exchange therapy disclosed that apparent half life time of each factors were remarkably shortened. For example, T 1/2 of factor I, E, VE, XI, antithrombin EE (AT IE) and alpha-2 plasmin inhibitor(AP) were 4, 14.5, 1-5, 4.7, 15 and 2.6 hours, respectively. In crossed immunoelectrophoresis of AT III and AP, patient plasma showed abnormal peaks of each protein which were considered to be enzyme-inhibitor complexes. Plasminogen was not detected before therapy(0 % by S-2251 and less than 5 % by Rocket IEP) in spite of AP remaining (16 % by S-2251, 25 % by Rocket IEP) and FDP was elevated to 80 mcg/ml at 4 hours after exchange. These findings indicated that, in some case of FHF, FDP did not increase because of absence of plasminogen even if accompanied with DIC.
Diagnosis Of Deep Venous Thrombosis By Combined Non-Invasive Flow Studies And Coagulation Tests
Authors: Caprini J A , Watts D R , Franck C A , Crampton A R , Vagher J P , Mitchell J
Published: July 2018

The diagnostic accuracy of Doppler ultrasound, impedance plethysmography, activated thrombelastography (TEG), and fibrin split products (FSP) assays were compared to the results of ascending venography in 127 consecutive patients with suspected deep venous thrombosis (DVT). The combination of Doppler with impedance plethysmography was 96% accurate in predicting normal limbs and 83% accurate in identifying limbs harboring thrombi. This combination of flow tests was more sensitive in detecting clots at the popliteal level or above (15/17 88%) than in the calf veins (18/23 78%). The coagulation tests had a high percentage of false positive results (TEG - 39%; FSP - 52%). TEG and FSP combined were 79% (67/87) accurate in normal limbs; 72% (29/40) accurate in those with thrombosis. 0/87 patients had all four tests positive in the face of a normal venogram. 1/40 patients with thrombosis had normal results with all four tests. This patient only had calf clots.

These results suggest that the addition of blood tests to flow studies does improve sensitivity and specificity when all tests are either positive or negative. The presence of normal blood tests and flow studies completely excluded thrombi in this series.