bluuebunny/crossref_metadata_2025 huggingface dataset:
ENHANCEMENT AND INHIBITION OF THE ACTIVITY OF RECOMBINANT ANALOGS OF TISSUE PLASMINOGEN ACTIVATOR
Authors: Erickson L A , Bergum P W , Hubert E V , Theriault N Y , Rehberg E F , Palermo D P , deMunk G A W , Verheijen J H , Marotti K R
Published: August 2018

Both the affinity of tissue plasminogen activator (tPA) for fibrin, which is associated with enhancement of its activity, and its susceptibility to inhibition contribute to the ability of this molecule to initiate vascular fibrinolysis. Full-length tPA is thought to consist of five structural regions designated the finger (F), growth factor-like (G), kringle 1 (K1), kringle 2 (K2), and protease (P) domains. Previous studies have suggested that the interaction of tPA with fibrin is primarily dependent upon the presence of the F and K2 domains, with its inhibition minimally requiring an intact active site. We have generated analogs of tPA with which to study further the relationship between the structure and the function of these domains. Synthetic DNA molecules, encoding specific tPA analogs and containing unique restriction sites in the interdomain regions, were expressed in Chinese hamster ovary cells. Serum-free media conditioned by transfected cells were then analyzed for both tPA antigen and activity. Analogs studied thus far include the full-length molecule FGK1K2P, GK1K2P, FK1K2P, FGK2P, FGK1P, FGK2K1P, and FK2P. In each case, samples of conditioned medium contained from 100 to 200 ng of tPA-like material per ml. Based on active-site titration of immunopurified analogs with 3H-DFP and assessment of tPA activity in the presence of CNBr-digested fibrinogen, the specific activities of analogs containing the K2 domain adjacent to the P domain were similar to that of native full-length tPA. In contrast, the specific activities of analogs with the K2 domain deleted (FGK1P) or transposed (FGK2K1P) were significantly reduced, indicative of the decreased ability of the fibrinogen fragments to enhance their respective activities. The extent of neutralization of the activity of each analog by platelet-derived PA inhibitor appeared to be similar to that of full-length tPA, confirming that the P domain contains the primary site of interaction between tPA and inhibitor. These data suggest that not only the presence of but also the position of the K2 domain are important for proper expression of tPA's activity.
ADMINISTRATION OF TICLOPIDINE IMPEDES ADP-INDUCED ACTIVATION BUT DOES NOT INDUCE A THROMBASTHENIC STATE
Authors: Potevin F , Lecompte T , Lecrubier C , Samama M
Published: August 2018

The aim of this study was to reassess the pattern of aggregation and to investigate the platelet-fibrinogen interaction, in response to various agonists, following T. treatment: 250 mg b.i.d. for at least 1 week. Platelets were obtained from healthy volunteers (n=8) as well as from patients with cerebro-vascular disease (n=5). The regimen was able to induce the well-known and expected alterations of platelet behaviour in citrated PRP. Washed platelets were prepared according to MUSTARD et al. (1972). (1) In a first set of experiments it was shown that these alterations persisted after resuspension of twice-washed platelets, in the presence of fibrinogen (3.7 μM); with ADP there was a marked impairment of aggregation, quantified either as maximal change in light transmission (DTLmax), or as maximal velocity; after stimulation with 100 μM ADP there was still a 40% decrease in DTL max and an about 50-fold faster de-aggregation as compared to values obtained without T. treatment. PAF-acether (0.1 to 3 μM): decreased DTLmax mainly with the higher doses, persistent de-aggregation but unimpaired velocity. Collagen (from Stago, μg.ml−1 ): DTLmax was decreased in response to low concentrations only (0.3 and 0.6). Arachidonic acid (20, 50, 100 μM) -induced aggregation remained unchanged, but the concentrations of U 46,619 able to induce an at least 50% DTLmax was shifted from 0.05 to 0.1 or 1.0 μM. (2) The association of human fibrinogen with stimulated platelets was quantified by plotting velocity against concentration of exogenous fibrinogen (0.06, 0.23, 0.92, 3.7 μM). The dose-response curves were modified in the presence of ADP (5 μM) but not PAF (0.5 μM) . With selected low concentrations of collagen or arachidonic acid addition of fibrinogen is a prerequisite for aggregation; under these experimental conditions T. treatment only induced slight modifications of the responses. Fibrinogen-dependent aggregation (DTLmax) of platelets from T. treated patients in response to ADP (10 μM) or after pretreatment with alpha-chymotrypsin was 1/3 and 2/3 of control values respectively. Taken together our results do not support the induction of a functionally thrombasthenic-like state, but rather suggest a selective impairment of ADP-mediated platelet activation. Accordingly, the pattern of platelet behaviour observed under T. treatment can be mimicked by adding ATP (as ADP-antagonist) to normal platelets.
ACTIVATION OF HEPARIN COFACTOR II BY PHOSVITIN, A PHOSPHOGLYCO-PROTEIN, AND OTHER PHOSPHATE-CONTAINING POLYANIONS
Authors: Church F C , Treanor R E , Whinna H C
Published: August 2018

We are characterizing the specificity of the polyanion-binding domain of the heparin/dermatan sulfate-dependent plasma protease inhibitor, heparin cofactor II (HCII). Various phosphate-containing polyanions accelerate the HCII-catalyzed inhibition of thrombin (T). Phosvitin, a phosphoprotein, enhances the HCII/T reaction at 25°C and pH 8.0 with the apparent second-order rate constant value (K2) increasing from 5 × 104 M−1 min−1 (in the absence of phosvitin) to 8 x 10' M”1 min as phosvitin increased from 0.05 to 30 ug/ml and then decreases as phosvitin is increased above 30 ug/ml. Apparent dissociation constant values for phosvitin-HCII and phosvitin-T are 450 nM and 10 nM, respectively. Polynucleotides accelerate the HCII/T reaction and have the following specificity (concentrations examined from 1-200 ug/ml): poly(guanylic acid) >> poly(adeny-lic acid, guanylic acid) > poly(inosinic acid) > poly(guanylic acid, uridylic acid) > poly(uridylic acid) = poly(adenylic acid) > poly(cytidylic acid). Polyphosphate anions (phosphate chain length, n, ranging from 5-100) enhance the HCII/T reaction. When compared at an equimolar phosphate concentration (1 mM), the rate was saturated at n = 50 with a maximum V.2 of about 5 × 107 M−1 min−1. Ca2+ (or Mg2+)-phosvitin/polyphosphate anion complexes and salmon protamine-polynucleotide complexes have lost the ability to enhance the HCII/T reaction. Phospho-pyridoxylated-HCII (lysine modified), with greatly reduced heparin cofactor activity, has lost its accelerating effect with phosvitin, polynucleotides and the polyphosphate anions. None of the above mentioned polyphosphate-containing compounds are effective at accelerating either the HCII-catalyzed inhibition of chymotrypsin or the antithrombin Ill-catalyzed T reaction. Our results suggest that (i) HCII is activated by the multiple negative charges of phosphate polyanions but they alone are not sufficient; (ii) the effective phosphate polyanions must also possess a specific conformation for maximum activity; and (iii) the phosphoserine-containing protein, phosvitin, can serve as a "template" for HCII/T.
SYNTHESIS. PROCESSING AND SECRETION OF HUMAN FACTOR VIII IN MAMMALIAN CELLS: REQUIREMENT FOR VON WILLEBRAND FACTOR
Authors: Wasley Louise C , Dorner Andrew J , Kaufman Randal C
Published: August 2018

In the plasma factor VIII exists as a complex with von Willebrand factor (vWF). The cloning of the cDNA for factor VIII has provided the ability to develop mammalian cell lines which express high levels of factor VIII by using appropriatate expression plasmids and DNA cotransformation with selectable markers. We have studied the synthesis, processing, and secretion of factor VIII expressed in baby hamster kidney cells and in Chinese hamster ovary cells by 35S-methionine pulse and chase labeling and analysis by immunoprecipitation with specific antibodies which recognize the light and heavy chains of factor VIII. In both mammalian cell lines, factor VIII is synthesized as a primary translation product of 230 kDa. A significant amount remains within the endoplasmic reticulum in a stable complex with a glucose regulated protein of 78 kDa. The remainder traverses into the Golgi compartment where it is cleaved to the heavy and light chain forms. Very shortly thereafter the mature factor VIII appears in the conditioned media as the mature heavy and light chain species. Very little single chain factor VIII is secreted into the conditioned media. The accumulation of factor VIII in the conditioned media requires the presence of vWF factor. In the absence of vWF, the factor VIII appears as unassociated heavy and light chains which are rapidly degraded. Bovine, porcine, or human 3WF all effectively stabilize human factor VIII expressed in these rodent cell lines. These results suggest the presence of vWF promotes factor VIII chain association which stabilizes the factor VIII to proteolysis.
EFFECTS OF HEPARIN AND COUMARIN ON DEPOSITION OF FIBRIN, PLATELETS AND PLATELET THROMBI ON RABBIT AORTA SUBENDOTHELIUM EXPOSED TO FLOWING HUMAN BLOOD
Authors: Inauen W , Bombeli T , Baumgartner H R , Haeberli A , Straub P W
Published: August 2018

The effect of heparin and phenprocoumon on thrombogenesis induced by rabbit aorta subendothelium (SE) was investigated in 20 volunteers using an ex vivo perfusion system. Blood was drawn directly from an antecubital vein through an annular chamber with exposed SE at lOml/min flow rate (650sec−1 shear rate) for 5min. Following buffer perfusion for 15sec, the middle portion of SE was removed for plasmin digestion and adjacent segments were fixed and embedded for morphometric analysis. Perfusions were performed 20 min after i.v. injection of heparin 1000, 2500 and 5000 IU, respectively; and during the decline and steady-state of prothrombin activity during a 2 weeks treatment with phenprocoumon to target INR of 5.0.

The amount of fibrin attached to SE, as measured by fragment E RIA in plasmin digests, correlated negatively with the dose of heparin (r=−0.83, P<0.001, n=48) and with INR during coumarin intake (r=−0.58, P<0.01, n=40). After high doses of either heparin or coumarin fibrin deposition on SE was virtually abolished (table). Platelet adhesion was increased. Platelet thrombus volumes and heights were reduced by heparin and coumarin.

We conclude that both heparin and coumarin dose-dependently inhibit fibrin formation induced by SE. In addition, both drugs impair platelet thrombus growth and/or stability indicating that these processes may also depend on the coagulation mechanism.
RESPONSE OF PROTEIN C AND PROTEIN S INHIBITORS IN LONG-TERM ORAL ANTICOAGULANT THERAPY
Authors: Mukhlova Montiel M , Bussey H
Published: August 2018

Protein C (PC) and its coenzyme Protein S (PS) are physiologic inhibitors of activated factors Va and Villa. Deficiency of either one of these inhibitors has been associated with venous thrombosis. Their activity is dependent on vitamin K for hepatic gamma carboxylation and it is depressed during oral anticoagulant therapy. Because rebound thrombosis complicates cessation of anticoagulant therapy, we investigated the response of PC and PS during long term oral anti coagulation. The study encompassed 30 patients ranging between 26 and 76 years of age, who have received therapeutic doses of coumadin from 15 days to more than 8 years. The conditions for which treatment was initiated were deep vein thrombosis, cerebral vascular accidents and cardiac valve replacements.

Factor VII and X activity was assayed by one step routine clotting assays. PC antigen (ag), total PSag and free PSag were assayed by Laurel 1 Rocket electroimmunodiffusion method. The measurement of the free PS was carried out after precipitation of C4b-binding protein with polyethylene glycol. PC activity was measured by clotting assay using PC deficient plasma to which was added patient plasma as a source of PC. Control group of 30 individuals in similar age group were assayed parallel with the patient samples. Compared with the control group the coumadin-treated patients showed substantial decrease of all factors studied. Statistical regression analysis of the coumadin group showed a significant increase in PS free (p = 0.014)during long term anti coagulation, while all of the other variables did not change significantly.

PCag and total PSag were decreased and their activities, as expected, were more severely affected. The ratio of PC activity to PCag averages 0.39 (normal >0.80) and free PS represented only 27% of the total PSag (normal about 40%). The inhibitors' persistent activity parallels that of the depression of Factors VII and X and there appears to be a balanced coagulation-inhibi-tion system. If PC and PS play a role in rebound thrombosis after a prolonged anticoagulation therapy, the changes may occur after discontinuation of medication.
CALIBRATED PLASMA PROCEDURE AND INR FOR PT STANDARDIZATION. DATA FROM THE FRENCH ETAL0N0RME QUALITY CONTROL SURVEYS
Authors: GOGUEL A , HOUBOUYAN A , ROUSSI J
Published: August 2018

One of the aim of the survey conducted in last december 1986 was to assess the efficacy of 2 procedures of standardization :

1) the INR system, derived from thromboplastin calibration and adopted in 1983 by the WHO.

2) the Reference Calibrated Plasmas (RCP) procedure, evaluated on large scale, through French interlaboratory trials (1977-85), exhibiting net improvement of the dispersion of overall data.

Labs were asked to perform with their local thromboplastin and method, the PT of a human lyophilized plasma 86 H/I, originated from long term antivitarnines-K (AVK) treated patients. Results were expressed *in time ; *in % activity, according to the traditional procedure based on saline dilutions of normal plasma ; *in INR using the ISI of the local reagent calibrated by the manufacturer. Calibrated plasmas procedure allow the determination of corrected activity ; *in % activity and INR, according to the linear calibration curve obtained from the PT of 2 reference calibrated plasmas with determinated activities in INR and % activity. These RCP were provided with and tested under the same conditions as plasma 86 H/I6 (2 systems of RCP : AVK and artificially depleted).

Statistical analysis shows that the "RCP" procedure leads to the best improvement of the interlaboratory variation for the overall data, and the best uniformization of mean results, whatever the way of expression (%, INR), the thromboplastin brand, and the method of PT testing. Results play also in favour of a system of AVK reference plasmas, giving a better grouping than the artificial calibrated plasmas. The INR system nevertheless provides a common scale of data reporting, but might hold profit from an efficient procedure of standardization, such as the calibrated AVK plasmas procedure.

Coefficient of variation (CV) expressed in %. Overall data PT of 86 H/I. French Etalonorme Survey.
DEVELOPMENT OF A SPECIFIC RADIODMMUNOASSAY FOR DETERMINATION OF PEPTIDES DERIVED FROM HUMAN LEUKOCYTE ELASTASE DEGRADATION OF HUMAN FIBRIN (OGEN)
Authors: Wallin R , Saldeen T
Published: August 2018

This paper describes a RIA for determination of the vasoactive peptide BB 30-43 and related peptides derived from leukocyte elastase degradaticn of human fibrin(ogen). The peptide was synthesized and could easily be labelled with 125I.Rabbits were iirmunized with BB 30-43 conjugated to bovine albumin. The antibody was found to bind about 5C% of the tracer in absence of BB 30-43 in a ˜1/800 diluticn. The RIA can detect peptide concentrations between 50 - 25000 pmol/L. The crossreaction with fibrinogen is very low (<0.001%) and with plas-min derived fibrin(ogen) peptides Bβ 1-42 and BB 15-42 also low (<0.2%). Plasma samples can be analyzed without any pretreatment. In an in vitro study fibrin and fibrincgen was degraded with plasmin or leukocyte elastase. Plasmin degradaticn of fibrin and fibrincgen did not release peptides which cross-reacted with our antibody, whereas leukocyte elastase degradation released peptides from both fibrin and fibrinogen which crossreact whith the antibody.

The imnunolqgical activity was not changed after degrading peptide Bβ 30-43 with a) trypsin, b) plasmin, c) batraxobin, d) thrombin, e) elastase, at +37°,1 h, in a molar ratio of 1:100. Even degradaticn by elastase (1:3.5) +37°, 1 h, did not destroy the iirmunological activity.

The imriunolcgical stability of peptide B< 30-43 in EDTA-plasma (+37°) seems to be very good. In citrated and heparinized plasma the activity of this peptide seems to vanish quite fast. In spite of these results we have detected high levels of iirmunolcgical activitiy in citrated or heparinized patient plasma. The molecular distribution of the peptides detected in plasma by our RIA corresponded to a fragnent containing about 25 amino acids. This fragnent seemes to be rather stable in plasma. When this fragnent was degraded with elastase in vitro a peptide with a molecular size resembling BB 30-43 was obtained. Over 300 patient samples have been studied. About 20 per cent were positive and the highest levels were found in patients with ARDS, septicaemia, severe renal failure, pulmonary embolism, pneumonia and pulmonary congestion.
ASSOCIATION OF VINCULIN TO THE PLATELET CYTOSKELET0N DURING RELEASE REACTION
Authors: Horvåth A , Asyee G M , Sturk A , Muszbek L
Published: August 2018

Vinculin is an Mr 130 kD protein, which in various cell types is located at the membrane attachment site of microfilaments and has been implicated in membrane-cytoskeleton interaction. Though we have previously verified its presence in platelets and showed that in resting platelets it is localized submembra-neously and around the ±-granules, its relation to the cytos-keleton is still to be elucidated. It has been also revealed by biochemical studies that in resting bovine gel filtered platelets vinculin is not cytoskeletal component, however about 50% of the total vinculin content became incorporated into the cytoskeleton during thrombin activation. To establish if its association to the cytoskeleton occured during pseudopode formation, aggregation or release reaction, cytoskeletons were prepared from platelets activated in various conditions and the time course of vinculin incorporation was analyzed by immuno-blotting. When pseudopode formation was inhibited by cytochalasin B pretreatment but neither aggregation nor release reaction induced by thrombin was prevented, the amount of vinculin in the Triton insoluble residue even increased. Phorbol myristate acetate (PMA), which induces pseudopode but not contractile gel formation, is a strong stimulus for aggregation, but induces only a slight release of the granule content. In parallel, only a low amount of vinculin was recovered in the cytoskeletal fraction following PMA induced aggregation. In contrast, when release reaction elicited by thrombin occured in the absence of aggregation the association of vinculin did not diminished.

The results clearly demonstrate, that platelet shape change and pseudopode formation are not prerequisites of the incorporation of vinculin into the cytoskeleton, which occurs parallel to the release reaction.
PLATELET EXTERNAL SURFACE MEMBRANE IS OSMOTICALLY DOUBLED IRRESPECTIVE OF SIZE OR SPECIES (HUMAN/BOVINE): DYNAMICS AND MEMBRANE SOURCES
Authors: Frojmovic Mony M , Wong Truman , Wylie Jane , White J G
Published: August 2018

Osmotic swelling can double the external plasma membrane surface area of human platelets independently of size, proposed to recruit the open surface-connected canalicular system ((SCCS) (Thrombos. Res. Suppl. VI: 119, 1986). As bovine (B) platelets have been reported to lack SCCS, we compared osmotic swelling for B and human (H) cells. Addition of water to piatelet-rich-plasma (10-90% v/v) caused sequential shape change and osmotic spherocyte (OS) formation, analyzed for size and surface area changes from time-dependent phase-contrast videomicroscopic images. Selected samples were fixed and stained with tannic acid prior to osmic acid fixation for visualization of open SCCS by transmission electron microscopy. B platelets required 3-4x less water dilution of PRP than H platelets, with significant OS forming at 20% water addition. Continued water dilution converted 50% of platelets to OS, with maximally stable swelling and no significant lysis for bovine OS up to 60% dilution. Electron micrographs of unactivated discocytes (D) and of optimally-swollen OS showed open SCCS in human D not detectable in any of the swollen platelets, though granules, mitochondria and a small number of vesicles and vacuoles persisted; no evidence for any open SCCS was found for bovine D or OS, though the OS otherwise appeared similar to H-0S. Geometric measurements of D and nonlysed OS showed a stable, maximal 2.1±0.1 fold increase in external plasma membrane surface area with osmotic swelling, identical for different-sized H platelets (mean volume = 2.8-6.8 f1) or for B platelets (3.6 f1 ). B platelets show equal or greater sensitivity for ADP-induced activation as H platelets, with 2-fold slower maximal rates of recruitment in early aggregation. As osmotic swelling appears to primarily externalize SCCS in H platelets, the identical relative amounts of internal membrane externalized for B platelets is hypothesized to arise from an osmotically more labile, “closed”, and structurally simpler SCCS or from a distinct membrane source tnan in H platelets.
PLATELETS EXPRESS A MEMBRANE PROTEIN COMPLEX IMMUNOLOGICALLY RELATED TO THE FIBROBLAST FIBRONECTIN RECEPTOR
Authors: Giancotti F G , Languino L R , Zanetti A , Grignani G , Tarone G , Dejana E
Published: August 2018

The heterodimer complex GpIIb-IIIa on human platelets can specifically bind fibronectin (FN) only when platelets are activated by thrombin. However unstimulated platelets can adhere and spread on a FN substratum. This suggests the existence of a second binding site for FN on the platelet surface that does not require activation for its expression. We have previously identified and characterized a membrane glycoprotein complex (Gp 150/135) that functions as fibronectin receptor (FN-R) in mouse fibroblast adhesion. To investigate whether this molecule was also present in platelets we have produced an affinity purified polyclonal antibodies monospecific for the lower subunit of the fibroblast FN-R. These antibodies specifically stained human and rat platelet surface as determined by fluorescence flow cytometric analysis and reacted with a component of 138 Kd m w in Western blot of platelet membranes. Experiments of differential extraction revealed that the 138 Kd component is an integral membrane protein. Moreover the antibodies precipitated the 138 Kd component together with a 160 Kd protein suggesting that the two molecules are associated in a supramolecular complex. A comparative analysis indicated that this protein complex is clearly distinct from the GpIIb-IIIa. In addition platelets from a thrombastenic patient reacted normally with 138 Kd but not with GpIIb-IIIa antibodies by Western blot analysis. These data indicate that normal human platelets express both GpIIb-IIIa and FN-R on their membrane and that these receptors are composed of structurally and antigenically distinct proteins.
HUMAN PLATELET GLYCOPROTEIN Ic-IIa IS AN ACTIVATION-INDEPENDENT FIBRONECTIN RECEPTOR
Authors: Piotrowicz Randolph S , Yamada Kenneth M , Kunicki Kunicki J
Published: August 2018

Human platelets express the membrane glycoprotein (GP) heterodimer GPIIb-IIIa, which functions as an activation-dependent fibronectin (Fn) receptor. We have immunopurified the components of an activation-independent Fn receptor (FR) from human platelets employing a well-characterized rabbit polyclonal antibody raised against the beta chain of the chicken embryo fibroblast (CEF) FR (anti-band 3). This antibody crossreacts with antigen(s) expressed on both chicken thrombocytes and human platelets and inhibits the binding of both normal and thrombasthenic platelets (lacking GPIIb-IIIa) to Fn-coated surfaces in the absence of platelet activation.

A monoclonal antibody directed against GPIIb-IIIa (AP2) partially inhibits the adhesion of normal platelets to Fn, but the combination of AP2 and anti-band 3 results in a level of inhibition greater than that obtained with either antibody alone. Thus, the presence of the FR alone is sufficient for the observed normal to enhanced binding of thrombasthenic platelets to Fn, whereas adhesion of normal platelets involves the synergistic action of the FR and GPIIb-IIIa. The adhesion of platelets to Fn mediated by the FR is inhibited by the tetrapeptide RGDS.

Immunopurified FR appears to be a complex of two proteins: an alpha chain with an apparent molecular weight of 155/130 KD (nonreduced/reduced) and a beta chain with an apparent molecular weight of 125/147 KD. The alpha chain is composed of two subunits, dissociated by reduction, with electrophoretic mobilities identical to platelet glycoproteins previously designated lea and IcB. The beta chain comigrates with that platelet glycoprotein known as GPIIa. In an immunoblot assay, anti-band 3 binds to GPIIa but not to GPIc. The fact that anti-band 3 iramunoprecipitates both GP therefore suggests that they exist in a complex.

Our findings establish GPIc-IIa as yet another platelet glycoprotein receptor complex and pave the way for future studies of the relative role of GPIIb-IIIa and GPIc-IIa in the adhesion of platelets to physiologic surfaces.
EXTRINSIC PATHWAY INHIBITOR (EPI):A SENSITIVE CHROMOGENIC SUBSTRATE ASSAY DEMONSTRATES THE RELEASE OF EPI TO THE BLOOD AFTER INJECTION OF HEPARIN
Authors: Abildgaard U , Sandset P M , Pettersen M , Hultin M
Published: August 2018

Control of the extrinsic pathway has been ascribed to negative feedback or to plasma inhibitors. Sanders et al recently showed that the inhibitor in plasma cooperates with F X in the inhibition of the tissue thromboplastin (TP)-F Vila complex (Blood 1985, _66,204). Our chromogenic substrate (CS) assay for the plasma inhibitors (Dahl PE et al, Thromb Haemost 1982, 48,253) was manipulated and the results confirm the cooperative effect of F Xa and EPI. When 1 ul of heated plasma was incubated with To (1/100 dil) and amounts of F VII and F X similar to tnose in 1 ul of normal plasma, progressive inhibition of TP-F VII was observed. Using higher amount of F X, the activation to F Xa dominated over inhibition of TP-F Vila. With optimal amounts (0.0005 U F VII, 0.0012 U of F X)in a volume of 250 ul, a 50% inhibition of TP-F Vila developed in 10 minutes. Remaining TP-F Vila was determined by adding 0.02 U of F X and, after 10 minutes, the CS S-2222. The A405 value is inversely related to EPI activity of test plasma or plasma fraction. Prior to assay, functional F II, F VII, F IX and F X in test plasma must be abolished, either by BaSo4 adsorption or by heating to 56° for 15 minutes. BaSo4 removes about 35% of EPI, heating about 15%. Antithrombin and heparin effects in the assay are prevented by specific antibodies and polybrene. EPI activity is reduced by phosphlipase C, suggesting lipoprotein nature. EPI is not released by venous occlusion, but following injection of heparin, EPI increases to 150-250% of base values. High EPI values are found in plasma from patients on heparin. We suggest that this increase in EPI contributes to the antithrombotic effect of heparin. Gel filtration sepa rates plasma EPI into 3 distinct peaks. The macromo-lecular peak accounts for more than half the activity in normal plasma. The activity of this peak increases markedly following heparin injection.
DEVELOPMENT OF A COAGULATION FACTOR X CONCENTRATE AS A BY-PRODUCT OF COAGULATION FACTOR IX PRODUCTION
Authors: Miekka Shirley I , Clark David B , Menache Doris
Published: August 2018

The American Red Cross is developing a Coagu.lation Factor X (FX) concentrate to provide a safer alternative for replacement therapy in Factor X deficient patients, who can experience thromboembolic complications with current treatments. Based on a survey of hemophilia treatment centers, we estimate the frequency of the homozygous disorder to be approximately 1/150th that of hemophilia A, or about 65 patients in the USA. We have devised a method for producing FX as a by-product of our Coagulation Factor IX concentrate (FIX). The method starts with adsorption of cryoprecipitate supernatant plasma with DEAE-Sephadex resin followed by elution of Vitamin K-dependent coagulation factors. This material is adsorbed to sulfated dejctran resin and Factors II and X are eluted by increasing the salt concentration. At 0.45 M NaCl, FII elutes quickly while FX is retarded and can be recovered essentially free of FIX by collecting the slower eluting material. FIX is then recovered at still higher ionic strength. The pooled FX is concentrated, diafiltered and treated to inactivate viruses. Approximately 30% of plasma FX was recovered in pilot scale experiments (600 liters plasma). Specific activity was > 51 FX units / mg protein corresponding to a purity of around 50% and 3000-fold purification over plasma. The ratios of Factors "X : II : IX : Protein C were 1.0 : <0.03 : <0.03 : 0.2. The major contaminant, conprising nearly 50% of the protein, was found to be inter-alpha trypsin inhibitor (IaI), a serine protease inhibitor whose function in plasma has not yet been determined. This inhibitor is also present in the DEAE-Sephadex eluate and. in the FIX concentrate. However, Western blot and HPLC analyses have shown that IaI is present in two different forms.

In FX it behaves as expected for the IaI monomer (Mr = 160 kDa), while in the DEAE-eluate and in FIX it exists also in r higher molecular weight form (≥400 kDa) corresponding either to aggregates, complexes or larger native species not previously described. The nature of the possible interaction of Ial w.vdi these coagulation factors is unknown and is currently boinn evaluated.
PREPARATION OF HIGH SPECIFIC ACTIVITY PLASMA AHF BY ANTI-FVIIIc IMMUNOAFFINITY CHROMATOGRAPHY
Authors: GRIFFITH M , LIU S A , NESLUND G , TSANG I , LETTELIER D , BERKEBILE R
Published: August 2018

To reduce the risk of pathogenic virus transmission associated with the therapeutic administration of plasma derived AHF (FVIIIc) we have developed a process wherein anti-FVIIIc immunoaffinity chromatography is used to isolate FVIIIc from a cryoprecipitate solution which has been treated with an organic solvent/detergent mixture to inactivate 1-ipid-enveloped viruses. A final ion exchange chromatography step is used to further remove contaminants (eg. anti-FVIIIc antibody) eluted with FVIIIc during the immunoaffinity step. FVIIIc obtained in the process has a specific activity of 1500 to 2000 AHF units (one stage clotting assay) per mg of protein, representing a ≥120,000-fold purification from plasma. The purified FVIIIc is stabilized for lyophilization and storage by the addition of human albumin. Polyclonal anti-FVIIIc immunoblots reveal polypeptides with apparent mol wts between 90,000 and 230,000 (heavy chain) and 70,000 to 76,000 (light chain). The major လcontaminant” in the preparation is von Willebrand Factor, one mol (Mr = 250,000 subunit) per mol of FVIIIc. Fibrinogen and fibronectin are barely detectable in the final preparation at levels of 1.0 and 0.3 ng per AHF unit respectively, representing a 3 million-fold purification of FVIIIc relative to these proteins. Anti-FVIIIc antibody, used in the immunoaffinity step of the process, is not detectable in the preparation at levels of 2.0 ng/mL (i.e. ≤0.01 ng per AHF unit). The extent to which virus reduction is associated with the process was also evaluated. The addition of an organic solvent/detergent mixture to cryoprecipitate solution resulted in HIV (and other lipid-enveloped viruses) inactivation to levels below the level of assay sensitivity in <5 min; representing a measurable 4 to 5 log reduction in virus titer. In addition, substantial (nonenveloped and lipid-enveloped) virus reduction was associated with the immunoaffinity step used in the process (3,400 to 50,000-fold depending on the virus/experimental conditions studie). The overall process results in a high specific activity AHF preparation which also appears to be substantially improved over previous AHF preparations with respect to viral contamination.
THROMBOSIS IN PATIENTS WITH HEPARIN-INDUCED THROMBOCYTOPENIA
Authors: Chesney C M , Lands R H
Published: August 2018

Heparin induced thrombocytopenia was studied in a 2000 bed teaching hospital with 21,000 admissions annually. Approximately 25% (5250 patients) received some form of heparin therapy (lV,SC, catheter flushes). In l6 months 21 patients(<0.4%) developed well-documented thrombocytopenia while receiving therapeutic bovine lung heparin(IV). In 12 cases thrombocytopenia appeared to be directly related to heparin (Group 1). In 9 cases additional disease processes were also present (Group 2). While on heparin 5 patients in Group 1 (42%) developed new thrombotic episodes including pulmonary embolus, transient ischemic attack, stroke, lower extremity embolus, and myocardial infarction. None of these complications resulted in death. All patients were tested by a sensitive and specific lUC-serotonin release method for detecting heparin-induced thrombocytopenia (Blood 67:27, 1986). The test was positive in 10/12 patients (83%) in Group 1 and only 4/9 (44%) in Group 2. All 5 patients with thrombosis had a positive test. In all 21 cases heparin was discontinued. Platelet count began to improve in 1-9 days and eventually returned to normal in all Group 1 patients and the 4 Group 2 patients with positive test. An additional patient not included in Group 1 or 2 received minidose heparin(8000 units SC q8h) as a prophylactic measure following cholcystectomy. Ten days later while still receiving heparin she developed a cold, pulseless right lower extremity. Test for heparin associated platelet antibody was strongly positive despite platelet count of 200,000 per ul. However review of medical record revealed platelet count of 433,000 the preceding day. Therefore the drop represents a decrease in platelet count of 54%. Embolectomy of the right femoral-popliteal system revealed white platelet thrombus. Heparin was discontinued.

The results presented here demonstrate that the l4C-serotonin release test is sensitive and specific for heparin induced thrombocytopenia. Well documented heparin induced thrombocytopenia occurs infrequently. However, thrombosis is not rare in these patients and may occur in the absence of absolute thrombocytopenia. Increased awareness of this entity and more accurate diagnosis may result in decreased mortality and morbidity.
CONGENITAL MACROTHROMBOCYTOPENIA, LEUCOCYTE INCLUSIONS, DEAFNESS AND PROTEINURIA: FUNCTIONAL AND ELECTR0NMICROSCOPIC OBSERVATIONS ON PLATELETS AND MEGAKARYOCYTES
Authors: Blockmans D , Heynen M J , Vermylen J , Verwilghen R
Published: August 2018

We report here a female patient of 33 years with a variant of Alport's syndrome (macrothrombocytopenia, leucocyte inclusions, deafness and proteinuria). The bleeding problems consisted of ecchymoses and menorrhagia, the deafness was of the ^ensorineural type. The platelet count in whole blood was 14.109/I, the mean platelet volume 22.8 μm3 . The template bleeding time exceeded 30 minutes. Ultrastructural studies of the peripheral blood revealed giant spheroid platelets with a high density of organelles, an abundance of vacuoles and an apparently disorganized microtubular system. In addition, unusual granule free areas were observed in the neutrophils of the patient and her mother. Granulocyte function was normal, except for a low myeloperoxidase content.

Functional studies of the platelets in platelet rich plasma showed normal aggregation curves related to the low platelet number, although no shape change could be elicited. Platelet aggregation studies in whole blood (impedance method) gave supernormal aggregation curves; this suggests the limited usefulness of this technique in patients with such large platelets.

The bone marrow contained numerous dysplastic megakaryocytes. In the mature granular megakaryocytes vacuoles and cysternae were organized in a radiating pattern demarkating elongated platelet territories. The platelet producing megakaryocytes showed fragmentation of the central zone and discharge of platelets through openings of the peripheral zone. These megakaryocytes had an immunological phenotype resembling that of very young cells (TR 14%, GP IIa 17% and GP IIIa 7%). The conversion of the elongated platelet territories into giant spheroid platelets probably results from remodelling within the circulation. The internalisation of plasma membranes would give rise to the extended invaginated canalicular system. Further studies are needed to explain the exact pathogenesis of this syndrome.
ARTIFICIAL EXON SHUFFLING: CONSTRUCTION OF HYBRID cDNAS CONTAINING DOMAINS OF TISSUE-TYPE PLASMINOGEN ACTIVATOR (T-PA) AND UROKINASE (u-PA)
Authors: de Vries C J M , Veerman N K , Pannekoek H
Published: August 2018

The intriguing finding that functions of t-PA coincide with structural domains and that these domains occur in related proteins, has been the basis to construct hybrid proteins by artificial exon shuffling to prove the conservation of functions in the shuffled domains. The heavy chain (Hch) of t-PA mediates both binding to fibrin and stimulation of plasminogen activator activity via its Finger- and Kringle-2 domain, whereas the light chain (Lch) contains the serine protease moiety of the protein. The Hch of u-PA is very homologous to the Lch of t-PA, but exhibits a higher plasminogen activator activity. This activity of u-PA is not stimulated by fibrin. We employed the ‘M13 in vitro outlooping’ technique to fuse the Hch of t-PA cDNA and the Hch of u-PA cDNA, to create two different hybrid cDNAs. On one hybrid cDNA, the t-PA and the u-PA sequences are coupled precisely at the exon-intron boundaries of the corresponding genes, while the other hybrid cDNA lacks a u-PA segment at the junction, encoding 13 amino acids of u-PA. The hybrid cDNAs were transiently expressed in mouse Ltk- cells and the recombinant proteins were characterized. The plasminogen activator activity of these proteins was determined in an indirect amidolytic assay, using plasminogen and the chromogenic substrate S2251. As anticipated, the activity of both t-PA/u-PA hybrid proteins is stimulated by fibrin, however, not to the same extent as t-PA. Remarkably, we found a decreased inhibition of the hybrid proteins by the endothelial plasminogen activator inhibitor (PAI-1) as compared to t-PA and u-PA, although stable complexes between the hybrid proteins and the inhibitor are formed. We conclude that functions of structural domains are maintained during exon shuffling
THE MITOGENIC EFFECT OF 15-HETE ON ENDOTHELIAL CELLS IS MEDIATED VIA DIGLYCERIDE KINASE INHIBITION
Authors: Setty B N Y , Stuart M J
Published: August 2018

We have previously demonstrated that 15-HETE, a mitogen for fetal bovine aortic endothelial cells (FBAECs), caused an accumulation of diglyceride (DG). Stimulation of DNA synthesis by 0AG (a synthetic DG analog) suggested that this mitogenic effect was mediated via elevation of cellular DG levels. In the present study we have delineated the mechanism by which 15-HETE causes DG accumulation by evaluating the effects of 15-HETE on the phosphati-dylinositol (PI) cycle. In [3H]inositol labeled cells, 15-HETE caused a decrease in PI content under both basal conditions (27%) and following A23187 stimulation (23%). The accumulation of DG and the decrease in PI suggested activation of phosphilipase C. However, no effect of 15-HETE was found on endothelial cell phos-pholipase C. A modulatory effect of 15-HETE on PI resynthesis was next evaluated using [3H] inositol. In 5 experiments, 15-HETE (30μM) inhibited the synthesis of PI in both unstimulated (51+15%, 1SD, P<0.001) and A23187 stimulated cells (80±19%, P<0.001). DG accumulation and inhibition of PI synthesis suggested a 15-HETE effect on the conversion of DG to phosphatidic acid (PA) via DG kinase. Using endothelial cell membranes as a source of this enzyme, formation of PA was nonlinear with time, suggesting an interference by PA phosphatase on PA formation. Human red cell membranes were therefore used in lieu of FBAECs as a source of DG kinase. In this system, production of PA was inhibited by 15-HETE in a concentration-dependent manner (IC50 of 22μM). This is the first report of the presence of a DG kinase inhibitor of biological origin. Our studies also delineate the mechanism by which 15-HETE exerts its mitogenic effect on endothelial cells.
13-HYDROXY-9,11-OCTADECADIENOIC ACID (13-HOD) INCREASES PROSTACYCLIN PRODUCTION IN ENDOTHELIAL CELLS
Authors: Setty B N Y , Berger M , Stuart M J
Published: August 2018

Recently, endothelial cells (ECs) have been shown to generate a potent vascular chemorepellant factor. This metabolite, 13-HOD is reported to be the major lipoxygenase product produced in microgram amounts in ECs (JBC 260:16056, 1985). We have studied the effect of 13-HOD on EC arachidonic acid (AA) metabolism, and report modulation of both AA release and conversion to prostacyclin. Using fetal bovine aortic ECs, 13-HOD stimulated prostacyclin production (RIA for 6KPGF1α ) by 40±13% (1SE), and 51±09% at 10 and 30μM (P<0.05; n=5). When the time-course of this effect was evaluated, 13-HOD (30μM) significantly enhanced the time-dependent release of 6KPGF1α by 31 to 51% between 5 and 120 min. (P<0.05 to 0.01; n=5). In [14C]AA labeled cells, this compound modulated both AA release and its subsequent conversion. In 5 paired experiments, 13-HOD (30μM) enhanced the release of AA from membrane phospholipids (9065±0553 cpm/well in controls vs 10738±1725 in 13-HOD treated cells; PC0.01). Analysis of cellular phospholipids revealed a significant decrease in [14C]phosphatidylethanolamine (62312±3963 cpm/well in controls vs 56959±4104 in 13-HOD treated cells; P<0.02). No significant changes were seen in the levels of phosphatidyl-choline, -serine, -inositol, or phosphatidic acid. Production of [14C]prostacyclin was stimulated by 56±16% (P<0.01 ), while total cyclooxygenase metabolites increased by 28±8% (P<0.01), suggesting effects on both cyclooxygenase and prostacyclin synthetase. 13-HOD, the major vascular product of linoleic acid enhances both AA release and metabolism, thus demonstrating an intimate hemostatic interaction between the metabolic products of these two polyunsaturated fatty acids in endothelial cells.
THE EFFECT OF ADRENALINE AND ISOPRENALINE ON INTRA-SPLENIC PLATELET KINETICS
Authors: Wadenvik H , Denfors I , Kutti J
Published: August 2018

It is well recognized that adrenaline decreases and isoprenaline increases the splenic pooling of platelets. However, the action of these adrenergic drugs on the splenic pooling mechanism is still unclear. The aim of the present study was to investigate the effect of an adrenaline and an isoprenaline infusion on intrasplenic platelet kinetics.

Methods. 13 healthy male volunteers were recruited for the study. Autologous platelets were labelled with 111-Indium-oxine. 6 of the subjects received an isoprenaline infusion (0.03 ug/kg/min) over 30 min, 4 an adrenaline infusion (0.2 ug/kg/min) and 3 an adrenaline infusion (0.1 ug/kg/min) over 40 min. The splenic blood flow (SBF), intrasplenic platelet transit time (PTT) and splenic platelet pool size (SPP) were determined twice by compartmental analysis of the equilibration of radiolabelled platelets between circulating blood and splenic platelet pool: at baseline and during the infusions. The initial distribution of injected radiolabelled platelets, between blood and splenic platelet pool, was followed dynamically with a computer assisted gamma camera. Student’s t-test for paired data was employed for comparison of mean values.

Comments. The adrenaline infusion diminished the exchangeable splenic platelet pool size by lowering the splenic blood flow. In contrast, the isoprenaline infusion increased the splenic pooling of platelets by increasing the splenic blood flow.
IN VIVO BEHAVIOR OF DETERGENT SOLUBILIZED PURIFIED RABBIT THROMBOMODULIN INJECTED INTO RABBITS
Authors: Ehrlich H J , Bang N U , Esmon N L , Esmon C T
Published: August 2018

Thrombomodulin (TM) is a thrombin (T) endothelial cell membrane receptor; the TM-T complex readily activates protein C resulting in anticoagulant activity. We investigated the biological effects of detergent solubilized purified rabbit TM upon i.v. injection into rabbits. 125I-labelled TM (lactoperoxidase method) disappeared from the circulation to a t 1/2 of 2.5 h.

The administration to rabbits of 2 doses of 50 μg/kg endotoxin 24 h apart did not accelerate the turnover rate of TM. In vitro tests demonstrated that .27 nM TM was required to significantly prolong the activated partial thromboplastin time (APTT) and .54 nM to significantly prolong the thrombin clotting time (TCT). At 3.78 nM TM, both the APTT and TCT prolonged to infinity while the prothrombin time (PT) was minimally affected. When TM was injected into rabbits in doses of 100, 200 and 500 μg/ml APTT and TCT immediately prolonged in a dose dependent fashion whereas no effect on the PT was observed. The disappearance rate of anticoagulant activity (prolongation of the APTT and TCT) closely paralleled the disappearance rate of 125I-labelled TM; t 1/2 for anticoagulant activity being 2.0 h irrespective of the dose administered. Thus, detergent solubilized purified rabbit TM survives in the circulation for appreciable time periods. While in the circulation TM is capable of binding T thereby prolonging the TCT as well as activating protein C thereby causing degradation of factors Va and VIIIa resulting in a prolongation of the APTT. Therefore, TM administered i.v. results in an anticoagulant effect of surprisingly long duration.
NO EVIDENCE OF HEPATITIS OR HIV TRANSMISSION IN VIRGIN HAEMOPHILIC BOYS TREATED WITH BRITISH HEAT TREATED FACTOR VIII CONCENTRATE (8Y)
Authors: Pasi K J , Hill F G H
Published: August 2018

HIV and hepatitis virus transmission is of major concern with factor VIII therapy. Non-A, non-B hepatitis (NANBH) has a near 100% incidence in patients previously treated with unheated large pool factor VIII concentrates. NHS heated high purity factor VIII concentrate (8Y) undergoes severe protracted heat treatment of the freeze dried concentrate theoretically sufficient to inactivate hepatitis viruses as well as HIV. Infusions of 22 different batches of 8Y have been given to 18 children with haemophilia A (10 virgin patients; 8 who had only received single donor cryoprecipitate) have been treated for up to 18 months. Regular testing for viral antibody seroconversion and biochemical liver enzymes have been made. None had had clinical or biochemical evidence of liver disease prior to the commencement of 8Y therapy. All these boys were immunized with HBVax at the time of the first treatment with 8Y and were HIV antibody negative.

Liver function tests were to be performed monthly but due to patient non-compliance this was only achieved in 60% of patients.

All patients receiving 8Y have remained anti-HIV seronegative. Only the virgin patients can be considered suitable for evaluation with regard to the transmission of NANBH. These boys by this time have received multiple batches of 8Y (mean 5 batches, range 1 to 14). In only 1 patient has an isolated rise in aspartate transaminase (AST) been noted (AST 27 to 131 IU/1) 6 weeks aftertreatment with a new batch, but no rise in alanine transaminase (ALT) or clinical evidence of liver disease was found. Viral serology was performed. AST returned to normal within 12 days.

This batch was received by 3 of the virgin and 2 of the previously cryoprecipitate treated boys. All these 5 boys who were exposed to the suspect batch had normal liver enzyme levels when measured within 4-6 weeks of exposure.

Of the 10 virgin patients receiving multiple batches of 8Y a transient rise in AST but with no rise in ALT has only been noted in 1 patient. In the absence of firm biochemical evidence of liver disease NANBH is an unlikely cause. Lack of transaminase rises in other virgin patients strengthens this assunption. We conclude that 8Y reduces the incidence of NANBH and HIV transmission.