bluuebunny/crossref_metadata_2025 huggingface dataset:
Further Evidence For The Role Of Platelet Surface-Associated Fibronectin In The Stimulation By Collagen
Authors: Bensusan H B , Holderbaum D , Khor K G , Orlando M
Published: July 2018

We have previously published evidence showing that 1) fibronectin (FN) can be extracted from intact platelets,
2) collagen fibers preincubated with plasma FN do not stimulate platelets, and 3) antibodies to human plasma FN stimulate platelets. We now present evidence that platelet stimulation by anti-human plasma FN antibodies depends upon antigen-antibody compatibility in platelets from individual donors. Antibodies to human plasma FN produced in rabbit and goat were purified on affinity columns of FN. Platelets from some donors were stimulated by antibodies from both species, some by one but not the other, and some by neither. These results suggest that there is a genetic variability of human platelet associated FN, presumably residing in the carbohydrate side chains. For this reason, donor-antibody compatibility must be considered in studies of platelet-membrane FN using antibodies. F(ab’)2 fragments of goat antibody caused stimulation of platelets from compatible donors. These data show that the stimulation of platelets is not due to any form of immune complex, which requires the Fc portion of the antibody. The Fab’ fragment caused no stimulation of platelets. However, when varying amounts of these fragments were preincubated with platelets, a dose-dependent inhibition of stimulation by collagen was seen. These data clearly indicate a role of platelet- membrane associated FN in the stimulation of the platelet by collagen. Three lines of monospecific antibodies were derived from hybridoma-induced mouse tumor ascites. Two of the antibodies from these lines stimulated the platelets from all donors while the other stimulated none. Furthermore, the plasma FN from all donors was shown to be antigenic to all antibodies of the goat whether or not the antibody stimulated their platelets. This fact, together with the observation that one monoclonal antibody caused no response, suggests that a portion of the platelet-membrane associated FN molecule is somehow masked or buried.
A Life-Long And Ultimately Fatal Bleeding Syndrome Due To Grossly Increased Levels Of Plasma Plasminogen Activator
Authors: Bennett B , Booth N A , Cumming A M , Cook I A , Dawson A A , Knox J
Published: July 2018

Episodes of haemorrhage due to increased fibrinolytic activity alone are uncommon and lifelong bleeding disorders due to excessive fibrinolysis are very rare. We report on a 46 year old male with a lifelong history of abnormal haemorrhage, usually occurring a day or two after minor trauma, surgery or dental extraction; no benefit had been derived from transfusion of fresh frozen plasma. There was no family history of bleeding but his father and grandfather had died at an early age of myocardial infarction and the patient had an abnormal lipid profile. No deficiency of any known clotting factor was present apart from a mild reduction of plasma fibrinogen (200mg/100ml). Platelet function was normal. The whole blood clot lysis time was consistently very rapid at 6-8 hours over a 2 year period of study. Levels of plasminogen, α1-antiplasmin, α2-antitrypsin, α2-macroglobulin, antithrombin III and CIs - inhibitor were normal but levels of plasminogen activator were grossly increased as were those of fibrin/fibrinogen related antigens (FRA).

He ultimately developed a massive spontaneous intracerebral haemorrhage which proved fatal after an illness lasting 7 days, during which administration of tranexamic acid abolished evidence of overactive fibrinolysis and resulted in a rapid rise of plasma fibrinogen to 700mg/100ml and a fall in FRA levels. Autopsy showed generalised severe atheroma. Thus the presence of grossly increased levels of fibrinolytic activity over many years did not prevent the development of atheroma in this patient in the presence of disordered lipoprotein metabolism though it may have contributed to the absence of thrombotic occlusions adjacent to atheromatous plaques.
Primary Structure Of α2-Macroglobulin
Authors: Sottrup-Jensen L , Stepanik T M , Lønblad P , Rider D M , Petersen T E , Magnusson S
Published: July 2018

The α2M tetramer of Mr 725,000 consists of four apparently identical single polypeptide chain monomers of Mr 180,000, corresponding to about 1450 amino acid residues. At the present stage of our sequence determination 1383 residues can be accounted for in three segments of continuous sequence namely the N-terminal (residues 1-440); the “middle” of 298; the C-terminal of approx. 645 residues. The eight oligosaccharide prosthetic groups are glucosamine-based and attached to Asn-residues, five in the N- terminal, three in the C-terminal segment. 25 CNBr-frag- ments have been found, 22-Met-residues have been overlapped. 23 Cys-sequences have been determined so far and ten -S-S- bridges identified. The Cys-residue (approx, no. 472 from the C-terminal in each chain) of the sequence is thiol-ester linked to the γ-carboxyl of the Glx-residue in native α2M which has not been reacted with proteinase or “inactivated” with methylamine or by heat inactivation.

Neither extensive internal sequence homology, nor homology with other proteinase inhibitors of known primary structure has yet been observed.
Factor Va And Platelet Cytoskeletons
Authors: Tuszynski G P , Walsh P N , Koshy A , Piperno J , White B
Published: July 2018

We have recently shown that the triton X-100 insoluble cytoskeletons from thrombin-activated platelets contain coagulation Factor Va (Tuszynski, G.P. et al. J. Cell. Biol. 87, 219, 1980). Factor Va activity was measured by two independent techniques, a one-stage clotting assay, and a two- stage chromogenic assay utilizing purified Factor Xa, prothrombin, and the chromogenic substrate S-2238. Further analysis of these cytoskeletons by SDS-PAGE and by immunodiffusion revealed the presence of fibrin. These findings suggest that a common mechanism may link alpha granular proteins to the platelet cytoskeleton. To investigate this mechanism, platelet cytoskeletons prepared from platelets treated under various conditions were assayed for Factor Va activity. Both the rate of Factor Va appearance and the final level of activity associated with the cytoskeleton were diminished 30-50% in platelets that were treated with aspirin (500μM) or with indomethacin (20μM). When secretion of Factor V, serotonin and nucleotides was inhibited greater than 90% by incubation of a platelet suspension with 2-de- oxyglucose (30μM), gluconolactone (40μM) and antimycin-A (9μM), cytoskeletons prepared from this platelet suspension contained less than 10% Factor Va activity of controls. The rate of appearance of cytoskeletal Factor Va activity increased with increasing thrombin concentration while the final level remained constant. Calcium ionophore, A-23187 (1μM), released Factor V but less than 2% was associated with the cytoskeleton suggesting that Factor Va, the activated from of Factor V, was required for binding. We conclude that prior secretion of Factor V and its further activation by thrombin are necessary for the association of coagulation Factor Va with the cytoskeleton. These results are consistent with the hypothesis that released Factor Va becomes associated with the cytoskeleton via cell surface components.
Human Plasma And Platelet Factor V Levels As Measured By Radioimmunoassay
Authors: Tracy P B , Peterson J M , Nesheim M E , Katzmann J A , Mann K G
Published: July 2018

Highly purified human Factor V was used for the development of a competitive double antibody radioimmunoassay (RIA) using 125I-human Factor V, burro anti-human Factor V antisera as the primary antibody and goat anti-burro antisera as the precipitating antibody. The standard curve allows the detection of as little as 20 ng of Factor V per ml of plasma. With this specific RIA for human Factor V, we have measured the level of Factor V in the plasma and platelets of normal individuals. The normal level of Factor V in plasma ranges from 4 to 14 μg per ml, with the average value equal to 7.0 ± 2.0 μg/ml (n = 64; 33 females; 31 males; 22 to 61 years of age). There appeared to be no correlation between antigen levels and age or sex. Factor V clotting assays were consistent with the RIA data for any given plasma preparation providing freshly drawn plasma was used in the bioassay. The bioassay data were quantitated based upon the specific activity of purified plasma Factor V; 1.7 units of Factor V equals 1 μg of protein. Plasma Factor V antigen levels were not affected by lyophilization of the plasma, prolonged storage of the plasma at -20°C or intentional conversion of the plasma to serum. The levels of Factor V present in washed human platelets were also determined using the RIA. Assay of washed platelets lysed in 0.2% Triton X-100 indicated that 0.6 to 0.85 μg of Factor V was present per 2.5 × 108 platelets (4400 to 6200 molecules of Factor V per platelet). This result is in marked contrast to our observations for the bovine system, where we found that bovine platelets possess approximately 400 to 800 molecules of Factor V per platelet, and plasma Factor V levels range from 30 to 50 μg per ml. In the bovine system, the platelets possess approximately 1% of the total Factor V present, while in human blood, the platelets possess as much as 10 to 15% of the total Factor V present.
Inhibition Of Human Factor Xa By Various Plasma Protease Inhibitors
Authors: Scully M F , Ellis V , MacGregor I R , Kakkar V V
Published: July 2018

The second order rate constants for the interaction of human factor Xa with antithrombin III (in the absence of heparin) α1 antitrypsin and α2 macroglobulin have been determined at pH 7.4 (50mM Tris HCl, 0.1M NaCl) in the presence of 3mM CaCl2. Factor Xa (free of activation peptide) and α1 antitrypsin were purified from outdated human plasma. Pure human preparations of antithrombin III and α2 macroglobulin were obtained elsewhere. The rate of inhibition was measured by determining the residual factor Xa activity at time intervals using S2337. In the case of the inhibition by α2 macroglobulin, measurement was made of bound factor Xa by carrying out the assay in the presence of soybean trypsin inhibitor, The rates were determined at molar concentrations of inhibitor to factor Xa of 1:1, 2:1, 4:1, and 8:1 and were concentration dependent indicating second order kinetcis. Suitable plots according to the full second order rate equation yielded straight lines from which the rates constants were calculated.

Calculated values for the inhibition of factor Xa by antithrombin III, α1 antitrypsin and α2 macroglobulin were 5.80×104 M-1 min-1, 1.36×104 M-1 min-1 and 4.00×104 M-1 min-1 respectively. The potential effectiveness of the inhibition for the inactivation of factor Xa in plasma can be calculated from these rate constants and the plasma levels of the inhibitors. Assuming plasma concentrations of the inhibitors to be 4.46μM, 42.4μM and 3.11μM respectively, the relative order of effectiveness under plasma conditions is α1 antitrypsin > antithrombin III > α2 macroglobulin in the proportions 4.64 : 2.08 : 1.00. These results are in contrast to the calculated effectiveness in plasma of these inhibitors against thrombin (of which antithrombin III is the most effective inhibitor). Cephalin was found to inhibit the interaction between factor Xa (2.8×104 M-1 min-1 at 100 μg/ml) and antithrombin III but not between factor Xa and α1 antitrypsin.
An Assessment Of Subcutaneous Ancrod In Prevention Of Deep Vein Thrombosis After Surgery For Hip Replacement
Authors: Belch J , Meek D , Lowe G , Drummond M , Campbell A , Forbes C , Prentice C
Published: July 2018

There is a high incidence of deep vein thrombosis (DVT) and pulmonary embolism (PTE) amongst patients undergoing surgery for hip replacement. We have assessed the use of ancrod (Arvin), a de- fibrinating enzyme in the prophylaxis of DVT.

In a randomised double-blind controlled trial 35 patients received daily subcutaneous injections of ancrod after operation for hip replacement and 38 patients received saline injections. DVT was detected by bilateral ascending venography (68 patients) or post-mortem (1 patient) 7-19 days after surgery. The frequency of major femoral DVT (> 5 cm long) was significantly reduced from 18 thrombi in the limbs of the placebo group to 5 in the ancrod group ( p<0.05). Major bilateral femoral DVT were similarly reduced from 7 to 1 patients by treatment with ancrod.

The overall frequency of all thrombi, including calf DVT, was however not significantly different between the groups. 5 patients had evidence of PTE (3 placebo, 2 ancrod). 6 patients within the ancrod group had evidence of increased wound bleeding, compared with one placebo patient, but in only one patient was this considered severe enough to require cessation of ancrod injections. As the majority of PTE perhaps arise from large femoral thrombi it would seem that ancrod prophylaxis, by reducing thrombus size, could aid the reduction of PTE in this group of patients.
Randomized Clinical Trial Of Titrated Dose Subcutaneous Heparin Versus Warfarin In The Long-Term Treatment Of Patients With Venous Thrombosis
Authors: Hull R , Delmore T , Carter C , Hirsh J , Genton E , Turpie A , Sackett D , Gent M
Published: July 2018

We have reported previously that low-dose subcutaneous (sc) heparin is less effective than adjusted dose warfarin sodium in the long-term treatment of venous thrombosis as 9 of 35 patients (26%) receiving low dose sc heparin developed acute recurrent venous thromboembolism compared with none of 33 patients receiving warfarin (p=0.001).

Seven patients on warfarin suffered bleeding compared with none on low-dose sc heparin (p < 0.005). Using an identical study design, 101 patients with venographically confirmed acute deep vein thrombosis have entered a second trial reported here, comparing higher doses of sc heparin 12 hourly, titrated at the onset (by adjusting the mid interval PTT to 1½ to 2 times control), compared with adjusted dose warfarin (PT 1½ to 2 times control). All patients were treated with full doses of heparin for 14 days and then randomized to either treatment group for a 12 week course. The patients were followed in a special clinic and routinely screened with leg scanning and impedance plethysmography at 3 week intervals and on an emergency basis if they developed recurrent symptoms.

During long-term treatment no patient on warfarin or titrated sc heparin developed recurrent venous thromboembolism. On follow-up, after discontinuation of long-term therapy, 4 patients randomized to warfarin and 4 patients randomized to titrated sc heparin developed recurrent deep vein thrombosis confirmed by objective testing. One of 51 patients on titrated sc heparin suffered bleeding compared with 9 of 50 patients on warfarin (p < 0.02).

These results suggest that titrated sc heparin is as effective as warfarin for preventing recurrent venous thromboembolism and its use is associated with less bleeding than warfarin.
Factors Influencing Thrombin Digestion Of Human Platelet Thrombospondin
Authors: Lawler J W , Chao F C , Palek J
Published: July 2018

Exposure of human blood platelets to thrombin results in the rapid release of a 420,000 dalton glycoprotein, designated thrombospondin, which is comprised of three polypeptides of equivalent molecular weight which are cross- linked by disulfide bonds. When purified human platelet thrombospondin is exposed to thrombin (4 units/ml) or plasmin (2 units/ml) for prolonged periods of time, proteolytic cleavages occur which result in the removal of 10,000 dalton and 30,000 dalton polypeptides with a concomitant decrease in the apparent molecular weight of the thrombospondin chains (Lawler, J.W. and Slayter, H.S., submitted for publication). In contrast, when the supernatant from thrombin- treated platelets is exposed to additional thrombin (4 units/ml) the 30,000 dalton, but not the 10,000 dalton fragment is released from thrombospondin. Proteolytic release of the 10,000 dalton polypeptide was inhibited by calcium and a nondialyzable component of the supernatant preparations. To further characterize the nondialyzable component, the supernatant was subjected to heparin-Sepharose affinity chromatography. Stepwise elution with varying NaCl concentrations yields a non-affinity flow through peak, a low affinity peak (eluted with 0.45 M NaCl) and a high affinity peak (eluted with 2.0 M NaCl). In addition to thrombospondin, the low affinity peak contains a β-thromboglobulin like protein as judged by RIA, apparent molecular weight on SDS-polyacrylamide gel electrophoresis (7,500-9,000 dalton) and heparin affinity. After dialysis of the low affinity peak against 0.14 M NaCl, 15 mM Tris-HCl (pH 7.6), 2 mM CaCl2 containing 0.02% NaN3, thrombin treatment resulted in the proteolytic release of the 30,000 dalton polypeptide, but not the 10,000 dalton polypeptide, from thrombospondin. These results suggest that calcium and a low molecular weight platelet protein can affect the pattern of thrombin digestion of thrombospondin.
ADP Initiates Phosphorylation Of Platelet Myosin Light Chain (MLC), A 40,000 Dalton Protein (40P) And Actin Binding Protein (ADP) Independent Of Secretion
Authors: Gerrard J M , Carroll R C
Published: July 2018

The mechanism by which ADP initiates platelet shape change and granule centralization is unknown. Recent studies of platelet proteins have provided evidence that raised intracellular calcium produces its effect by promoting protein phosphorylation. We have studied the influence of ADP on the phosphorylation of platelet proteins in both platelet rich plasma (PRP) and washed platelets, using the incorporation of 32p to identify phosphorylated proteins. 10u M ADP was found to cause a 2-5 fold increased phosphorylation of MLC, 40P and ABP. Pretreatment of the platelets with 100uM ASA prevented secretion of serotonin but decreased the ADP induced phosphorylation only by about 1/3. 2u M ADP stirred with platelets in PRP caused reversible aggregation without secretion and was associated with increased phosphorylation of MLC, 40P and ABP. The results show that ADP can cause phosphorylation of platelet proteins independent of secretion. We suggest ADP raises intracellular calcium levels to cause the phosphorylations which initiate contractile protein interactions to produce shape change and granule centralization. The results also show that platelet granule secretion must be dependent on other factors in addition to phosphorylation of these proteins.
Correlation Of Tension Generation And Myosin Light Chain Phosphorylation In A Thrombin Activated Platelet Contractile Model
Authors: Daniel J , Sevy R , Salganicoff L
Published: July 2018

Previous experiments have established that a 20,000 daltons protein which becomes phosphorylated when platelets are stimulated by thrombin is a myosin light chain. A model of thrombin activated platelets, in which the tension generated by contraction of the platelet aggregate is measured under isometric conditions, was used to study the relationships between steady-state myosin light chain phosphorylation and tension generation. After thrombin activation the platelet aggregate contracted, and in the contracted state myosin light chain was found to be phosphorylated. Relaxation induced by either PGI2 (which activates adenyl cyclase) or 120 mM K2SO4 containing 2 mM EGTA (which is presumed to deplete cytoplasmic Ca++) was accompanied by dephosphorylation of myosin light chain. Recontraction of the relaxed preparation with either ADP (10-4M) or epinephrine (10-6M) in the presence of Ca++ was accompanied by rephosphorylation of myosin light chain. The relationships between isometric tension, cAMP production and myosin light chain phosphorylation and dephosphorylation were studied in the contracted thrombin activated preparation subsequently relaxed with graded doses of PGI2(3×10-9 to 3×10-6M), such that complete dose-response curves were obtained. There was a striking positive correlation between the level of isometric tension and the degree of phosphorylation of myosin light chain. Over the same dose range of PGI2 there was no increase in cAMP levels, within the error of the detection procedure, until the ED- 50 (approx. 3×10-8M) for relaxation with PGI2 was exceeded. At the same time that relaxation and myosin light chain dephosphorylation occurred in response to PGI2 there was phosphorylation of a 22,000 daltons band; relaxation with K2SO4-EGTA did not affect this band. The above experiments are consistent with the hypothesis that myosin light chain phosphorylation is directly involved in platelet contraction.
Acquired Platelet Storage Pool Disease In Chronic Autoimmune Thrombocytopenia (ITP) And Marrow Dyspoiesis
Authors: Malpass T W , Savage B , Slichter S , Harker L A
Published: July 2018

The mechanism underlying platelet dysfunction in patients with chronic ITP or marrow dyspoiesis has been characterized with respect to template bleeding times, platelet nucleotides and a granule contents. Ten patients with modest thrombocytopenia (93,000 plat/ul ± 53,000) due to chronic ITP (4), “preleukemia” (4), primary myelofibrosis (I), and polycythemia vera (I) were selected for study on the basis of discrepantly prolonged bleeding times in excess of that predicted by the platelet count, i.e. BT = 30.5 -platelet count/3850. Using high pressure liquid chromotography, adenosine diphosphate (ADP) and adenosine triphosphate (ATP) were measured in both whole platelet extracts and the supernatant of thrombin stimulated platelets. From these measurements it was possible to determine the relative distribution of ATP and ADP in the cytoplasmic and dense granule compartments. The α granule protein, platelet factor four (PF4), was determined by RIA.

Whereas dense granule ADP was markedly reduced (mean 0.92 ± 0.58 u moles/1011 platelets compared with 2.41 ± 0.20 in 20 normal subjects; p < 0.01), platelet cytoplasmic ATP and ADP were not decreased 3.49 ± 0.45 and 0.40 ± 0.27 u moles/1011 platelets compared with 2.73 ± 0.27 and 0.3l ± 0.15 in normals respectively). The severity of the platelet dysfunction (difference in the observed bleeding times from predicted) varied directly with the decrease in dense granule ADP (R2 = 0.88). Only three patients (all with dyspoiesis) also demonstrated an associated α gronule abnormality as shown by partial depletion of the platelet content of PF4. Platelet survival studies done simultaneously with 51Cr and 14C-serotonin showed discrepantly shortened 14C-serotonin disappearance in 3 patients (I ITP, 2 dyspoiesis) suggesting preferential dense granule losses from circulating platelets. Possible mechanisms of platelet dense granule ADP depletion may include activation and release or leak of dense granule contents or defective platelet development in the marrow. We conclude that platelet dysfunction may occur in some patients with chronic ITP and hematodyspoiesis due to acquired dense granule storage pool disease.
Effects Of A Monoclonal Antibody To Human Platelet Glycoprotein I On Platelet - Von Willebrand Factor Subendothelium Interactions
Authors: Ruan C , Tobelem G , McMichael A , Drouet L , Legrand Y , Caen J P
Published: July 2018

A monoclonal antibody (AN51) to human platelet glycoprotein I (GPI) secreted by a hybrid myeloma has been tested on platelet functions. AN51 bound to normal and thrombasthenic platelets while it failed to bind to platelets from 6 patients with Bernard-Soulier syndrome (BSS). The nature of the antigen recognized by AN51 was determined by demonstration that the antigen, which was chymotrypsin sensitive, gave a peak at 150,000 daltons on SDS-PAGE after immunoprecipitation. AN51 strongly inhibited Ristocetin, bovine factor VIII or porcine factor VIII induced aggregation but did not modify ADP, collagen type I or type III, thrombin or arachidonic acid induced aggregations. Furthermore the adhesion-aggregation of platelet induced by microfibrils was also inhibited by the antibody AN51. Platelet adhesion to untreated or collagenase treated rabbit aorta subendothelium, using the Baumgartner technique, was impaired by AN51, and the inhibition was more pronouced at high shear rate conditions. AN51 decreased the binding of 125I-factor VIII/Willebrand factor (FVIII/WF) to human platelets in presence of Ristocetin. The use of this monoclonal antibody directed against platelet GPI permits a better understanding of the platelet-platelet and platelet-subendothelium interactions mediated by FVIII/WF and will facilitate the purification of the platelet membrane glycoprotein lacking in BSS which may be the receptor for FVIII/WF.
Interactions Of Platelets With Proteins Of Plasma And Matrices
Authors: Lahav Judith , Hynes Richard O
Published: July 2018

We have been analyzing the interactions of human blood platelets with plasma proteins arid with proteins of extracellular matrices (collagen, fibronectin etc.) using a variety of techniques. Using a modified enzyme-linked immunosorbent assay (ELISA), as few as 5.103 bound platelets could be determined quantitatively and proteins bound from solutions could be detected. Using this method the interaction of the plasma proteins fibronectin, FVIII-VWF and fibrinogen with one another, with components of the basement membrane and with the blood platelet were studied. Antibody against carefully washed human platelets recognizes fibronectin, FVIII-VWF, and fibrinogen as well as platelet surfaces. However, specific antisera to these three proteins fail to bind to the surface of gel-filtered, unactivated platelets. When gel-filtered platelets adhere to plastic in the absence of plasma proteins, they spread. Such platelets do react with antibodies to fibronectin, FVIII-VWF, and fibrinogen. These results suggest that these three plasma proteins are found inside platelets but not on their surfaces prior to activation and that they become exposed upon spreading. Furthermore, these proteins are undetectable on the surfaces of unactivated platelets by surface iodination followed by immune precipitation. We have also studied the interactions of platelets with various collagen substrata by Immunofluorescence microscopy which shows recruitment of plasma fibronectin by platelets. These findings confirm and extend recent suggestions concerning the associations of fibronectin, FVIII-VWF and fibrinogen with human platelets. Finally, we are using chemical crosslinking reagents to analyze the same protein-protein and protein-platelet interactions.
Correlations Between Platelet Consumption And The Surface Chemistry Of Neutral Polymers In Baboons
Authors: Hanson S R , Ratner B D , Haffman A S , Harker L A
Published: July 2018

The rate of destruction of autologous 51Cr-labeled blood platelets has been measured in baboons having femoral arteriovenous shunts composed of various test materials. Platelet survival was normal in baboons with chronic Teflon-Silastic shunts alone demonstrating that the surgical variables had no detectable effect. Platelet survival data obtained when other materials were studied were analyzed by computer modeling to estimate both the rate of shunt-associated platelet destruction and the rate of platelet removal by ordinary senescent mechanisms. Rates of shunt associated platelet destruction, normalized with respect to shunt surface area (plats/cm2 -day), were used to assess material thrombogenicity. Polymers studied included poly(ethyl methacrylate) (EMA), poly(hydroxyethyl methacrylate) (HEMA) and two intermediate copolymers (HEMA/EMA: 1/1, 1/3) radiation grafted onto two substrates (silastic and polyethylene). Og both substrates rates of platelet consumption (range: 5-20 × 108 plats/cm2 -day) correlated with: I) HEMA/EMA monomer ratios; 2) graft polymer water absorption measurements; and 3) surface ratios of carbon to oxygen atoms and of various functional groups as determined from electron spectroscopy for chemical analysis (ESCA). With ten commercial polyurethane materials rates of platelet consumption (range: 2-23 × 108 plats/cm2 -day) correlated inversely with the percentage of surface carbon atoms forming hydrocarbon bonds, and directly with the surface content of ether-type linkages (which will be related to polyether “soft” segments) as determined from ESCA.

These observations in primates indicate that the thrombo- genecity of prosthetic materials is strongly influenced by their surface physicochemical properties.
The Influence Of Materials And Platelet Inhibition On The Thrombogenicity Of Arterial Grafts In Man
Authors: McCollum C N , Norcott H C , Hawker R J , Goldman M , Drolc Z , Snape E
Published: July 2018

Prosthetic arterial grafts often thrombose when used to bypass diseased small arteries due to the deposition of laminated platelet thrombus. The rate of lll-Indium labelled platelet accumulation on autogenous vein, polytetra- fluoroethylene (PTFE, Gore-Tex) and double velour Dacron (Microvel) has been investigated in patients and the influence of aspirin and dipyridamole (ASA/DPM) evaluated.

Two days before surgery 40 patients undergoing femoro-popliteal bypass were started randomly and double blind, on either ASA 300 mgm + DPM 75 mgm tds or placebo. One week postoperatively autologous 111-Indium labelled platelets were injected and isotope emissions over the graft and contralateral leg counted for 7 days. Graft thrombogenicity was calculated as the daily rise in the ratio of counts, graft/contralateral thigh.

Three placebo and one ASA/DPM prosthetic grafts occluded prior to study. Thrombogenicity (mean ± SEM) was greatest in the Dacron grafts at 0.22 ± 0.03 on placebo (n=7) and 0.16 ± 0.03 on ASA/DPM (n=5) (p < 0.05). The effect of therapy however, was most striking in reducing thrombogenicity of PTFE grafts from 0.17 ± 0.03 (n=4) to 0.06 ± 0.01 (n=7) (p < 0.02). The thrombogenicity of 0.03 ± 0.005 was so low in the 13 vein grafts that the effect of therapy could not be determined.

The 111-Indium platelet technique described may be used to quantitate in vivo platelet deposition. In man the combination of ASA/DPM reduced the rate of thrombus formation on prosthetic materials. PTFE grafts with ASA/DPM therapy most nearly approach the low thrombogenicity of vein.
Role Of Verapamil (Calcium Blocker) In The Reduction Of Platelet Deposition In Gore-Tex Grafts In Dogs
Authors: Fuster V , Dewanjee M K , Murphy k , Vlietstra R E , Didisheim P , Kaye M P
Published: July 2018

Platelet calcium appears to be essential in platelet activation. Accordingly, Verapamil (a channel calcium blocker) was tested “in vivo” as a possible platelet inhibitor agent. Fifteen dogs underwent bilateral femoral artery grafting with one polytetrafluoroethylene (Gore-Tex) and one autologous vein graft. Peroperatively, five dogs were infused continuously with Verapamil (6 μgs/kg/min) and 9 dogs were left untreated and the arterial prostheses were surgically implanted. Using autologous Indium-111-labeled platelets injected 24 hours prior to surgery as a platelet marker, grafts were removed 1 hour following resumption of blood flow. Radioactivity per unit weight of Gore-Tex, vein and blood were determined with a gamma counter and the relative radioactivity with respect to the internal reference standard of blood is tabulated below

Verapamil-treated dogs showed a significant reduction in the “in vivo” platelet deposition on Gore-Tex and autologous venous grafts. Preliminary data indicates that “in vitro” platelet aggregation with soluble calf skin collagen (515 μg/ml) and ADP (2-100 μm) is not different between control and drug-treated dogs.

Calcium channel blockers may be promising “in vivo” platelet inhibitor agents; in addition, they may provide further understanding of the different processes involved in “in vivo” and “in vitro” platelet activation.
Evaluation Of Pharmacologic Inhibitors Of Platelet Function In Baboons
Authors: Hanson S R , Harker L A
Published: July 2018

The ability of aspirin (ASA), dipyridamole (DIP), sulfinpyrazone (SP) and combinations of these agents to prevent arteriovenous cannula thromboembolism In baboons .has been assessed by measuring the destruction of autologous 51Cr-platelets. In 24 studies with untreated baboons (10-12 kg), thrombogenic polyurethane femoral A-V cannulap (Biomer, 50 cm, 4 mm i.d.) consumed 22.5 ± 4.5 × 108 plats/cm2 -day. In animals with identical shunts, the efficacy of various antithrombotic treatment regimens was determined by measuring the percent reduction in platelet consumption (PC) compared to untreated control values. All drugs were given to at least four animals by oral administration twice daily.

ASA, 3-100 mg/kg/day, did not significantly reduce PC (p >0.2). SP In doses of 10, 50 and 100 mg/kg/day reduced PC by 7, 48, and 97% respectively. DIP, 1, 2.5, 5, 10 mg/kg/day, reduced PC by 14, 34, 5l,and 99% and was significantly more effective than SP (p <10-5). ASA (10, 15, 20 mg/kg/day) given with DIP (2.5 mg/kg/day) reduced PC by 47, 67, and 94% demonstrating significant and dose dependent potentiation of DIP by ASA. ASA (10 mg/kg) potentiated DIP (2.5 mg/kg/day) less effectively when given only with the first dose of DIP, or when given twice daily but two hours after administration of DIP (p <0.01). In both cases PC was reduced about 67% (vs. 94% for simultaneous administration twice daily). DIP (2.5 mg/kg/day) plus SP (10, 20, 40 mg/kg/day) reduced PC by 50, 51, and 72% in an additive fashion without potentiation. Similarly SP (20 mg/kg/day) given with DIP (2.5 mg/kg/day) plus ASA (10 mg/kg/day) reduced PC by 65% In an additive manner.

It is concluded that PC Is more effectively reduced by DIP than either SP or ASA. DIP Is potentiated by ASA but not SP. DIP and ASA should be administered simultaneously in multiple daily doses.
Characterization Of Cdna Clones Coding For The α And β Chains Of Bovine Fibrinogen
Authors: Chung D W , Rixon M W , MacGillivray R T A , Davie E W
Published: July 2018

Radioactive cDNA was synthesized from a bovine liver mRNA preparation enriched for fibrinogen mRNA activity. This cDNA was used in a solution hybridization assay to screen a collection of recombinant plasmids carrying bovine cDNA inserts. A number of plasmids containing cDNAs for fibrinogen were identified.

The nucleotide sequence of the insert of one plasmid, designated pBIβ1, has been determined. It contains 1372 base pairs and codes for the C-terminal 424 amino acids of the β chain of bovine fibrinogen. This and other previous-ly reported partial sequences enabled the construction of the complete amino acid sequence of the bovine β chain. Comparison of this sequence with that of the human β chain shows a high degree of homology. The positions of all the cystine and tryptophan residues are identical.

The nucleotide sequence of the insert of another plasmid, designated pBIα1, has also been determined. This plasmid contains 965 base pairs and codes for the C-terminal 203 amino acids of the bovine a chain. This region shows only 46% homology with the corresponding region in the human α chain. A hypervariable region, consisting of 53 amino acids, is present in the bovine a chain. This region shows little homology with the corresponding region in the human α chain. Furthermore, it is 26 amino acids shorter than the corresponding region in the human a chain.

The cDNAs to the bovine fibrinogen chains will provide good probes for the study of human fibrinogen and the organization and structure of its genomic DNA.
Fibrinogen Lille : Aα7ASP ⤒ ASN
Authors: Morris S , Denninger M H , Finlayson J S , Menache D
Published: July 2018

Previous studies on fibrinogen Lille indicated a structural defect in the NH2-terminal region involving the Aα chain. The rate and extent of fibrinopeptide A release were greatly diminished. This was further evidenced by the NH2-terminal analysis of fibrin which showed alanine in addition to tyrosine and glycine and by the finding that fibrinopeptide A was being maintained on fragment E for a prolonged period of plasmic hydrolysis.

To further elucidate the abnormal structure, fibrinogen Lille was isolated by glycine precipitation, reduced, carboxymethylated and the chains separated by ion exchange chromatography. Approximately 50 nanomoles of either normal or Lille reduced and carboxymethylated Aa chains were subjected to automated sequence analysis using a single cleavage program in 0.1 M Quadrol. PTH amino acids were identified by a combination of high performance liquid chromatography and mass spectroscopy. The normal Aα chain was sequenced up to position 10 and the expected fibrinopeptide A sequence was obtained. Aa Lille chain was analysed up to position 20 on one sample and up to position 10 on another sample. A single sequence was obtained in both experiments; it was identical to the normal Aa sequence with the exception of position 7, which was identified as a mixture of aspartic acid and asparagine. These results, consistent with a single base change, suggest two distinct populations of fibrinogen and a substitution in the highly conserved region of fibrinopeptide.
A New Case Of Dysfibrinogenemia : Isolation Of The Abnormal, Unclottable Fibrinogen Population
Authors: Jandrot-Perrus M , Aurousseau M H , Josso F
Published: July 2018

In a 81-year-old healthy woman, gross abnormalities of fibrin formation in routine tests led to the discovery of a dysfibrinogenemia.

Abnormal and control fibrinogens were purified in parallel using precipitation by glycine (Kazal) ; final clottability was 95-98 % for the control and 50 % for the patient’s fibrinogen. Electrophoretic behaviour of the fibrinogen momecule, the three chains and the products of fibrin cross-linking by factor XIII a was normal. Functional studies gave the following results :
(i) delayed coagulation by thrombin, Reptilase and Venacil with gross abnormalities of the clot; (ii) inhibition of coagulation of normal fibrinogen ; (iii) poor fibrin monomer aggregation (opacimetry) ; (iv) delayed fibrinogen proteolysis by plasmin (SDS-PAGE) . Release of fibrinopeptide A by thrombin was incomplete (RIA).

Fibrinogen NH2-terminal residues were found normal, but the presence of ALA-residue in fibrin clot and in the supernatant showed that part of fibrinogen was not clotted, either copolymerized with fibrin or remaining in solution. Gel filtration of the supernatant showed the presence of both soluble complexes and fibrinogen characterized by the elution volume of the peak and NH2-terminal analysis. This fibrinogen population was unclottable by thrombin and inhibited clotting of normal fibrinogen.

These preliminary results suggest the existence of a defect on the A-a chain of this abnormal fibrinogen which was called fibrinogen Bondy.
A Study Of Microcrystals Of Fibrinogen
Authors: Tranqui L , Hewatt E , Wade R
Published: July 2018

We are interested in the application of image analysis to the structural determination of fibrinogen. This analysis is performed on the electron microscope images obtained from microcrystals of slightly degraded fibrinogen. The proteolysis of fibrinogen is carried out using bacterial proteases fixed onto sepharose. In our study 3 mg of sepharose-proteases are mixed with 16 mg of fibrinogen. Digestion-times of 30 min. to 4 h. are employed. The analysis of reduced fibrinogen lysates, by polyacrylamide gels in SDS,shows a progressive disappearance of the Aα chain, while lighter chains appeared with a MW of 30000. After 4 h. of digestion, the MW of the degraded fibrinogen is about 267.000, it clots after exposure to thrombin. As shown on unreduced samples on 5 % acrylamide gels in SDS, the digest is heterogeneous ; homogeneous samples are obtained by a separation on ion exchange-chromatography. From the crude fibrinogen lysates,several forms of microcrystals are obtained after a dialysis at low ionic strength (μ=0,005 ; pH = 6,2). Different forms of microcrystals are obtained from some fractions after chromatography. All these microcrystals are observed in the electron microscope after negative staining. The optical diffraction patterns obtained from these micrographs permit the selection of the most ordered regions ; thus we have established that the largest regions of ordered structures are obtained when the Aα chain of fibrinogen is degraded into a lighter chain of MW 30.000. One of the forms obtained from the crude fibrinogen digests has been studied in more details, it has a unit cell of 450 Å x 90 Å its optical diffraction shows a resolution of 30 ft. The microcrystals obtained from the fractions of chromatography have a unit cell of 440 Å/150 Å, their optical diffraction patterns show details to 35 Å. They have pgg projection symetry down the c-axis. The structures are examined after the elimination of the noise on a computer system. A first conclusion is that it is not evident that these images are simply related to the supposed elongated form of the fibrinogen molecule.
The High Affinity Calcium Ion Bending Sites of Fibrinogen
Authors: Ross Joan , Kemp Graham D
Published: July 2018

There is considerable evidence that fibrinogen contains a number of strongly bound calcium ions and these appear to have a significant role in the structure and properties of the molecule. Most of the evidence suggests that there are three such strongly bound calcium ions in fibrinogen and each of the two fragments D contains one of these. It has been suggested that the section of the (A) α chain which is the region of the molecule first attacked by plasnin is involved in binding calcium ions. Should this constitute the third site it follows that this calcium ion must link the two (A) α chains and the site may well be destroyed by minimal plasnin attack. The figure of three calcium ions bound, however, must be open to sane doubt due to the difficulty in evaluating data from Scatchard plots prepared from. a system, such as fibrinogen, which contains a number of identical ligands with more than one binding affinity. Accordingly we have developed methods to prepare fibrinogen in as intact a form as possible, and used such fibrinogen in flow dialysis systems. Studies of calcium ion release during proteolytic degradation of fibrinogen lead us to conclude that there are probably only two high affinity, calcium ion specific binding sites in fibrinogen.
Fibrin Membrane Endowed With Biological Function: Immobilization Of Enzymes And Living Cells
Authors: Inada Y , Hagiwara H , Saito Y , Matsushima A
Published: July 2018

Fibrin membrane formed from fibrinogen with thrombin and blood coagulation factor XIII was a superior matrix for preparing immobilized enzymes and living cells. Enzymes such as asparaginase, chloroplast ATPase or catalase and living cells such as Chlorella cells or sea urchin eggs were embedded in fibrin membrane without imparing their functions. The enzymes participating in degradation of purine bases(uric acid), uricase, allantoinase and allantoicase together with catalase were embedded simultaneously in matrices of fibrin molecules. The multienzyme complex thus prepared had an ability to degrade urate to glyoxylic acid and urea via allantoin and allantoic acid. Four enzymes in urea cycle, ornithine carbamoyltransferase, argininosuccinate synthetase, argininosuccinate lyase and orginase together with inorganic pyrophosphatase, were immobilized into matrices of fibrin molecules. The immobilized multienzyme system not only had an ability to carry out urea cycle continuously at least over several hours, but also had a greately improved efficiency over the corresponding soluble system. To the best of our knowledge this is the first report to show an immobilization of efficient cyclic enzyme system.
Further Observations On The Possible Association Of Human Platelet Membrane Glycoproteins IIb And IIIa
Authors: Rosa J-P , Pidard D , Kunicki T , Nurden A T
Published: July 2018

Studies are described which represent a continuation of our investigation into the role of membrane glycoproteins (GP’s) IIb and IIIa during human platelet aggregation. The surface proteins of washed platelets were labelled with 125I by the lactoperoxidase-catalysed method prior to membrane isolation by the glycerol lysis procedure. Solubilisation of the membrane proteins by triton X-100 was followed by their analysis by crossed immunoelectrophoresis (CIE) using a rabbit antibody prepared against normal human platelets. In the absence of divalent cation chelation GP IIb and Ilia were contained within a single 125I-labelled immunoprecipitate. When the isolated membranes were solubilised by triton X-100 in the presence of 5mM EDTA, GP IIb and IIIa formed distinctand separate immunoprecipitates during CIE. In order to further investigate this finding 125I-labelled membrane proteins solubilised by triton X-100 in the presence or absence of EDTA were subjected to centrifugation for 18 h at 100,000 g over a 10-40% sucrose gradient containing the nonionic detergent. The results confirmed that in the presence of divalent cations lib and Ilia were associated in a complex, and that this complex is dissociated by EDTA. The IgG..L is an alloantibody isolated from a polytransfused thrombasthenic patient that has been shown in previous studies to inhibit ADP-induced platelet aggregation and the binding of 125I-fibrinogen to normal human platelets in the presence of ADP. When the IgG..L was incorporated in an intermediate gel during CIE it was shown to precipitate the complex containing IIb/IIIa but under identical conditions it did not precipitate the individual glycoproteins dissociated by EDTA. Divalent cation-mediated changes in the orientation of lib and Ilia in the platelet membrane should be considered in assessing the role of these GP’s in platelet function.
Evidence For A Separate Localization Of Platelet Glycoprotein IIb And III In The α-Granule Membrane
Authors: Gogstad G O , Hagen I , Korsmo R , Solum N O
Published: July 2018

Characterization of a well defined human platelet α-granule preparation by crossed immunoelectrophoresis revealed the presence of an immunopre- cipitate which previously has been shown to contain GP IIb and GP IIIa. The corresponding antigen is known to be surface-located. Surface iodination of whole platelets followed by subcellular fractionation, crossed immunoelectrophoresis of the fractions and autoradiography of the immunplates revealed a considerable labelling of this antigen in the membrane fraction whereas no labelling was detected in the corresponding antigen in the α-granules. The surface membrane form seems to be immunologically identical to the α-granule form, however. Another surface membrane-located glycoprotein, glycocalicin-related protein, was present in the α-granules in only trace amounts indicating little contamination by plasma membranes. The presence of GP IIb and GP IIIa in the α-granules was confirmed by SDS-polyacrylamide gel electrophoresis. The antigen containing these glycoproteins was found to be a constituent of the α-granule membrane. We conclude that GP IIb and GP IIIa are constituents of the α-granule membrane as well as the plasma membrane in platelets.